A Method for Serial Tissue Processing and Parallel Analysis of Aberrant Crypt Morphology, Mucin Depletion, and Beta-Catenin Staining in an Experimental Model of Colon Carcinogenesis
© McGinley et al; licensee Springer 2010
Received: 11 March 2010
Accepted: 23 April 2010
Published: 18 May 2010
The use of architectural and morphological characteristics of cells for establishing prognostic indicators by which individual pathologies are assigned grade and stage is a well-accepted practice. Advances in automated micro- and macroscopic image acquisition and digital image analysis have created new opportunities in the field of prognostic assessment; but, one area in experimental pathology, animal models for colon cancer, has not taken advantage of these opportunities. This situation is primarily due to the methods available to evaluate the colon of the rodent for the presence of premalignant and malignant pathologies. We report a new method for the excision and processing of the entire colon of the rat and illustrate how this procedure permitted the quantitative assessment of aberrant crypt foci (ACF), a premalignant colon pathology, for characteristics consistent with progression to malignancy. ACF were detected by methylene blue staining and subjected to quantitative morphometric analysis. Colons were then restained with high iron diamine–alcian blue for assessment of mucin depletion using an image overlay to associate morphometric data with mucin depletion. The subsequent evaluation of ACF for beta-catenin staining is also demonstrated. The methods described are particularly relevant to the screening of compounds for cancer chemopreventive activity.
Keywordscolon carcinogenesis aberrant crypt foci mucin depletion beta-catenin image analysis morphometry
Visualization of aberrant crypt foci (ACF) in methylene blue-stained colon whole mounts was first described by Bird  and gave insight into early pathologic changes in colon epithelium associated with carcinogen treatment and the subsequent development of colon cancer. Morphologic criteria used to identify ACF include: darkly stained epithelium, slight elevation of epithelial cells above the surrounding normal mucosa, increased pericryptal zone, enlarged crypt size, and changes in crypt shape [1–4]. While numerous ACF can be found within colons of carcinogen-treated animals, tumor multiplicity is quite low with one or two carcinomas observed per animal . This situation has given rise to a controversy regarding the clinical relevance of ACF [3, 6, 7] since they have been widely used as a surrogate endpoint for colon carcinogenesis to screen compounds for cancer chemopreventive activity in preclinical models . Loss of mucin occurs in a small percentage of ACF, and these mucin-depleted foci (MDF) have been proposed to identify ACF with an increased probability of progressing to cancer, but this concept also has come under scrutiny [5, 9–12]. Herein, we describe methods and evaluate the usefulness of morphometric image analysis to yield insight into this problem.
Current approaches used to prepare and evaluate colon whole mounts for identification and characterization of ACF [1, 12–15] have a number of issues, which are not conducive to consistent acquisition of digital images and subsequent image analysis. They include: (1) tissues mounted on filter paper or cork boards to flatten during fixation must be removed from the object for transillumination and visualization of the stained mucosa [1, 16]; (2) high-quality image capture of free-floating stained specimens can be difficult or unfeasible due to specimen movement in fluid media and natural topographical changes present in the colonic tissue; (3) real-time ACF counting and scoring, using a dissecting microscope, are prone to errors from observer fatigue; (4) labeling and storage of free-floating tissue is problematic; and (5) comparative analysis of additional staining techniques is hindered by lack of consistent specimen orientation. These issues have impeded the development of a more complete understanding of ACF as a surrogate biomarker for colon cancer and may account, in part, for the apparent disparities that have been reported between the prevalence of ACF and subsequent development of colon carcinomas .
Based on our extensive work in developing techniques appropriate for preparing mammary gland whole mounts [17–19] and carrying out image analysis on digitally captured images of mammary tissue [20, 21], we postulated that whole mounts of colon could be prepared by directly adhering the tissue to glass microscope slides and the whole mounted tissue then be used for semi-automated acquisition of contiguous images of the entire colonic mucosa; this would circumvent many of the limitations listed above. Moreover, we undertook the development of approaches to permit methylene blue staining, image acquisition, re-staining with high iron diamine–alcian blue (HID-AB) and further image acquisition in order to address current questions about the relevance of ACF detected by methylene blue versus those in which mucin depletion can be detected as predictors of subsequent development of colon carcinomas [5, 9, 22–24]. For this purpose, methods were developed for the construction of non-destructive overlay images to permit sequential evaluation of ACF, detected in methylene blue images, for mucin depletion. Additional techniques for processing of whole mount preparations, paraffin-embedding, and microtomy were developed so that ACF and mucin-depleted foci could be further interrogated for the presence of dysplasia and for beta-catenin accumulation by immunohistochemical analysis, since beta-catenin accumulation is directly linked to colon cancer development [10, 23–25]. We report, in detail, the methods developed for these procedures and illustrate their use in the morphometric analysis of ACF.
2 Materials and Methods
Female Sprague Dawley rats were obtained from Taconic farms (Germantown, NY) at 20 days of age. At 21 days of age, rats were given an ip injection of 50 mg 1-methyl-1-nitrosourea/kg body weight (Ash Stevens, Detroit, MI) . Rats were group-housed in an environmentally controlled room maintained at 22°C and 50% relative humidity with a 12 h light–dark cycle. They were fed a modification of the AIN-93G diet formulation  and deionized water ad libitum. The study was terminated 9 weeks following carcinogen injection. The work followed ethical guidelines approved by the Colorado State University Animal Care and Use Committee.
2.1 Whole Mount Preparation
At necropsy, rats were skinned, and a midline incision was made in the lower abdominal wall to expose the internal organs. A second incision was made at the pubic symphysis, and the pelvic bones were spread apart to reveal organs at the base of the abdomen. Reproductive organs were removed in toto and the gastrointestinal tract exposed. The colon was cut at the anus, carefully lifted out of the abdomen and excised above the ileo-cecal valve to visually maintain the anatomical orientation of the tissue. Colon length was measured in millimeters from the anus to the cecum, and residual colonic mesentery was removed using Adson forceps (George Tiemann & Co, Hauppauge, NY, Cat. No. 105-235-1). The cecum, anus, and rectum were removed, and the colon was trisected starting at the distal end into three equal lengthwise segments that were operationally defined as representing descending, transverse, and ascending regions, respectively. The colon was briefly immersed in phosphate-buffered saline (PBS) pH 7.4, and hardened fecal pellets were removed using a gentle milking action of the colonic tissue between the index finger and thumb. A longitudinal incision running the entire length of the colon was made using a small pair of scissors (George Tiemann & Co, Cat. No. 105-411), which had been modified in house using a bench grinder to blunt and smooth both tips in order to prevent perforation of the colon and minimize disruption of the fragile colonic mucosa while making the incision. Large deposits of soft fecal material were removed using Adson forceps, and the tissue was washed in PBS to remove residual fecal material from the colonic mucosa.
The colon was laid serosa side down onto a Kimwipe® to wick away excess moisture. This process was repeated a few times by gently lifting the colon and placing it on a dry section of the Kimwipe® until the serosal surface became tacky, i.e., increased resistance noted when separating the colon from the Kimwipe®. Whole mounts of colonic tissue segments were prepared by laying the serosal surface down onto 75 × 25 mm 3-aminopropyltriethoxysilane-treated microscope slides (Statlab Medical Products, McKinney, TX, Cat. No. 418). Adson forceps were used to carefully stretch the four corners of the tissue toward the edges of the slide. Once tacked down at the corners, Adson forceps were used to stretch the areas lying in between by carefully grasping a small portion of underlying serosa at the edge of the colon and slightly lifting the tissue off the glass while pulling toward the edge of the slide, being careful not to damage the delicate mucosa nor introduce air bubbles between the tissue and glass. This process was quickly repeated along all four sides until the tissue was stretched completely flat across the surface of the slide. Whole mounts were exposed to air for a period of 4 min prior to immersion in fixative to prevent tissue detachment from the glass slide. Whole mounts were fixed in 10% neutral buffered formalin for 24 h, rinsed in tap water for 15 min, and stored in 70% ethanol.
2.2 Methylene Blue Staining and Image Acquisition
2.3 Digital Extraction of ACF
Composite digital images were opened in Photoshop® v9.0.1 (Adobe Systems, Inc., San Jose, CA) and saved as PSD files (Photoshop® native format with support for layers), one file for each of the three whole mount slide preparations per animal representing ascending, transverse, and descending colon. ACF were individually circumscribed on a separate transparent layer using the Photoshop® pencil tool with a contrasting bright color in conjunction with an Intuos 3 stylus and digital tablet (Wacom Technology Corp., Vancouver, WA). ACF were digitally excised from the image by selecting the transparent area outside the circumscribed borders using the magic wand tool and then applying the inverse command to select only the colored borders of the circumscribed ACF. The background layer was activated and that layer, via copy command, was used to create a new transparent layer containing only the excised ACF. The image was duplicated and flattened to remove all layers except the excised ACF on a white background. The resulting image was saved as a separate TIF file for image analysis.
2.4 HID-AB Staining
Methylene blue-stained colon whole mounts were stained with HID-AB as described by Caderni et al.  to look for evidence of mucin depletion in ACF. Images of HID-AB-stained colon whole mounts were acquired as outlined above and added to PSD files as a separate layer and aligned with the original methylene blue-stained images. Once aligned, the same layer used to circumscribe methylene blue-stained ACF was used to extract HID-AB-stained ACF as a separate layer using the technique described above, and the resulting image was saved as a separate TIF file for image analysis. The use of separate aligned layers permitted each image to be toggled on or off at will enabling qualitative assessment of the same ACF in both methylene blue and HID-AB stained whole mounts.
2.5 Image Analysis
The HID-AB macro used three different segmentation thresholds based on hue, saturation, and intensity to isolate areas within each ACF and place them into three separate classes based on color: HID (dark brown), AB (blue), and unstained (absence of brown or blue color). Class areas were measured and expressed as a percent of the total area for each ACF. Unstained areas, representing ≥85% of the total area of each ACF, were operationally defined as MDF. Morphometric data from both macros were exported via DDE to an Excel spreadsheet.
2.6 Whole Mount Tissue Processing, Paraffin-Embedding, and Microtomy
Colon whole mounts on glass slides were placed in Tissue Tek® plastic slide racks (VWR, West Chester, PA, Cat. No. 25608-868) and processed in an automatic tissue processor using an abbreviated processing schedule and infiltrated with molten paraffin. Whole mounts were bisected down the long axis, and each half was trisected yielding six pieces of tissue per slide; tissues were embedded as separate blocks, mucosa side down. Five-micron serial sections were cut from each block, mounted onto 3-aminopropyltriethoxysilane-treated glass microscope slides, and stained with hematoxylin and eosin (H&E) according to normal laboratory protocol. Images of H&E sections were acquired as mentioned above and added to the PSD file as a separate layer. This layer was aligned with previously captured methylene blue and HID-AB layers, thus allowing qualitative assessment of ACF across all three staining techniques.
2.7 β-catenin Immunohistochemistry
Sections were cut at 5 μm and mounted on 3-aminopropyltriethoxysilane-treated slides and heat-immobilized in a 60°C oven for 20 min. Sections were deparaffinized in three changes of xylene, hydrated in a series of graded ethanols, rinsed in deionized water followed by three rinses in Tris-buffered saline (TBS) [50 mM Tris–HCl, 150 mM NaCl, pH 7.6 with 0.05% Tween 20 (Dako, Carpinteria, CA, Cat. No. S1968 and S1966)]. Subsequent steps were carried out at room temperature using an Autostainer (Dako, Carpenteria, CA). Anti-β-catenin (BD Biosciences, San Jose, CA, Cat. No. 610153) 1:50 was applied and incubated for 1 h followed by two rinses in TBS. FITC donkey anti-mouse Fab'2 (Jackson ImmunoResearch Laboratories, West Grove, PA, Cat. No. 715-096-151) 1:100 in 10% normal donkey serum (Jackson ImmunoResearch Laboratories, Cat. No. 017-000-121) was applied and incubated for 30 min followed by two rinses in TBS. 4',6-diamidino-2-phenylindole (DAPI) (Invitrogen, Carlsbad, CA, Cat. No. D1306) 300 nM was applied and incubated for 10 min followed by two rinses in TBS. Slides were rinsed in two changes of deionized water for 1 min and allowed to air dry under a fume hood in the dark.
Images were acquired using a Zeiss Axiocam HRm camera (Carl Zeiss, Thornwood, NY) coupled to a Zeiss Axioskope II microscope (Carl Zeiss). Multi-channel acquisition within the Axiovison v4.1 software (Carl Zeiss) was used to obtain separate images at a magnification ×400 for both FITC (filter excitation and emission of 480 and 535 nm, respectively) and DAPI (filter excitation and emission of 350 nm and 460 nm, respectively) stained areas, which were rendered as a composite image containing both fluorophores.
2.8 Statistical Analyses
Descriptive statistics, ANOVA, and multivariate analysis of variance were performed using SYSTAT v12.02 (SYSTAT Software, Inc., Chicago, IL).
Excise the entire colon from cecum to anus as quickly as possible, i.e., <4 min from time of euthanasia.
Wet the serosal surface of the excised colon by quickly dipping in PBS pH 7.4
Remove hardened fecal pellets by gently milking the serosa between the index finger and thumb.
Make a longitudinal incision along the entire length of the colon using a pair of small, blunt-tipped scissors and remove any large deposits of soft fecal material.
Grasp the superior (proximal) end of the colon using a pair of Adson forceps (George Tiemann & Co, Hauppauge, NY, Cat. No. 105-235-1). Note the orientation of the mucosal surface and dip repeatedly in PBS until all visible fecal material is removed.
Remove the colon from PBS and lay the serosal side down on a Kimwipe®.
Trisect the colon into three equal length segments, representing ascending (proximal), transverse, and descending (distal) portions of the colon.
Place each segment on a separate Kimwipe®, maintaining orientation, and fold an unoccupied portion of the Kimwipe® on top of the mucosal surface to wick away excess PBS.
Unfold the Kimwipe®, grasp the superior end of the colon, and gently peel off the Kimwipe®, and lay it down on a used portion of the paper. Repeat this process until all excess PBS has been wicked away and the serosa becomes tacky.
Use Adson forceps to carefully transfer the segment from the Kimwipe® to a clean, 75 × 25-mm 3-aminopropyltriethoxysilane-treated microscope slide (Statlab Medical Products, McKinney, TX, Cat. No. 418), mucosal side up and orient the segment with the superior end toward the frosted label of the slide.
Use Adson forceps to gently grasp underneath the outer edge of the colon segment and pull toward the edge of the slide being careful not to disturb the fragile mucosa. Repeat this process all the way around the colon segment so that the serosal surface is mounted completely flat against the glass slide.
Allow the colon whole mount to air dry for 4 min to prevent detachment. Completely immerse the slide in 10% neutral buffered formalin and fix for 24 h. Rinse in tap water and store in 70% ethanol until ready to stain.
3.2 Methylene Blue Staining
Remove the slide(s) from 70% ethanol and rinse briefly in deionized water (dH2O) for 1 min. Do not stain more slides than can be imaged in a 2-h period.
Immerse the slide(s) in freshly prepared 0.05% methylene blue (Sigma, St. Louis, MO., Cat No. M9140) by dipping the slide 20× at 1-s intervals and then let sit undisturbed in stain for 3 min at room temperature (RT).
Rinse the slide(s) briefly in dH2O until all excess methlyene blue stain has been removed.
Remove one slide at a time from dH2O, capture images, and then store in 70% ethanol.
3.3 Circumscribing Methylene Blue-Stained ACF in Photoshop®
Open the captured image into Photoshop®, create a new layer, and label as "Outlines"
Click the Outlines layer to activate and use the Photoshop® pencil tool (pixel diameter = 2, hardness = 100%) with a strong contrasting color, e.g., red to completely circumscribe each visible ACF. A stylus and digital tablet will increase the speed and efficiency of this process while greatly reducing fatigue associated with using a mouse.
Once all ACF have been circumscribed, use the Photoshop® magic wand tool (tolerance = 30, continuous and anti-alias options checked), and click anywhere outside the circumscribed border of an ACF, i.e., in the blank portion of the Outlines layer.
Choose Select and Inverse from the Photoshop® menu.
Click the background layer to make it the active layer. Place the cursor inside the circumscribed boundary of any ACF, right click, and choose Layer via Copy. This will create a new transparent layer with ACF only. Label the layer as "methylene blue ACF" and save the image file in .PSD format.
Turn off all layers with the exception of the methylene blue ACF layer and save a copy of the file in .TIF format (no layers). The image is ready for image analysis.
3.4 High Iron Diamine–Alcian Blue Staining
Remove the slide(s) from 70% ethanol and rinse in dH2O for 5 min.
Immerse the slides in a small staining dish of diamine solution consisting of 480 mg of N'-N'-dimethyl-m-phenylene diamine (Sigma, Cat. No. D3886), 80 mg of N'-N'-dimethyl-p-phenylene diamine (Sigma, Cat. No. D4139), 200 ml of H2O, and 5.6 mL of 60% ferric chloride (Sigma, Cat. No. F2877). Allow slides to stain for 18 h at RT.
Rinse the slides 3× in dH2O.
Immerse slides in a freshly prepared, filtered (No. 1 Whatman) solution of 1% Alcian blue (Sigma, Cat. No. A3157) in 3% acetic acid and stain for 30 min at RT.
Rinse slides 3× in 80% ethanol followed by 3× in dH2O.
Capture images and store in 70% ethanol.
3.5 Circumscribing HID-AB-Stained ACF in Photoshop®
Open the original methylene blue-stained .PSD image in Photoshop®.
Open the HID-AB-stained image, and drag the layer to the original methylene blue-stained image, which will create a new layer in the original image file. Label the layer as HID-AB, and arrange the layer in the palette such that it lies directly above the methylene blue-stained layer.
Reduce the opacity of the HID-AB layer from 100% to 50% in order to make the layer semi-transparent.
Click on Edit and Free Transform from the Photoshop® menu and adjust the XY position of the HID-AB layer so that it is directly superimposed over the top of the methylene blue background layer, and all edges are aligned. Increase the opacity of the HID-AB to 100% once the layer is aligned.
Repeat steps 2–6 listed under method 3.3, "Circumscribing methylene blue-stained ACF in Photoshop®" (substituting "HID-AB ACF" for "methylene blue ACF") to create a new TIF image with only HID-AB-stained ACF. The image is ready for image analysis.
4 Results and Discussion
Advances in automated micro- and macroscopic image acquisition and digital image analysis have created the opportunity to investigate gastrointestinal disease processes, such as colon carcinogenesis and inflammatory bowel disease, with enhanced sensitivity and accuracy; however, a significant limitation in this field has been the limited application of quantitative morphometric approaches to the colon. Additionally, little detail has been reported in the literature on colon processing, and there is lack of uniformity in the methods that are described. To address these constraints, each step in the processing of the colon—excision, whole mount preparation, fixing, staining, processing, and histological or immunohistochemical evaluation—was evaluated and modified as appropriate. To address the issues related to this method's development, each phase is further discussed below.
Removal of the colon is associated with several problems which are mitigated by the following procedures. (1) The length of excised colon at the distal end can be influenced by variation among technicians in the excision process. Since a significant number of ACF are found in the descending colon [16, 29–32], it is imperative that this portion of colonic tissue be removed fully intact. The incision through the pubic symphysis and clearing of reproductive organs accommodates full colon excision to the anus. (2) The forceps used to excise the colon from the abdomen can induce crush artifact, visible in methylene blue-stained whole mounts. Manual removal of the colon with the fingers minimizes the introduction of excision artifacts. (3) Excised colon segments can be difficult to maintain in the proper orientation (i.e., proximal vs. distal). Excising the colon with the cecum attached permits accurate anatomical orientation of the tissue for purposes of measurement and subsequent preparation of ascending, transverse, and descending colon segments on slides.
4.2 Whole Mount Preparation
To avoid tissue detachment from the slide, debris removal (e.g., mucus, mesentery, excess peritoneal fluid, and fecal material) is critical. By doing this, the requisite connective tissue is exposed providing improved spreading of the colon on the glass slide and better adhesion of the serosal surface to the slide. Washing the colonic tissue in PBS must be followed by blotting of the serosal surface to increase tackiness of the tissue. A brief period of exposure to ambient air prior to immersion in fixative also enhanced attachment of the colonic tissue to the glass. Significant amounts of gouging artifacts can result at the periphery of tissue when Adson forceps are employed for stretching. A modified technique utilizing forceps to anchor the corners first then stretching the areas lying in between is advisable to prevent damage to colonic tissue at the edge. The whole mount preparation is a fundamentally different approach to analysis of colonic ACF, compared with current protocols in the literature, making extensive staining and image analysis procedures possible.
4.3 Methylene Blue Staining
Protocols for methylene blue staining to detect ACF vary by diluent (water or saline), methylene blue concentration, staining time, and agitation technique during staining. Various combinations of these approaches were evaluated for the staining of the glass whole mounted colons and marked variations in staining intensity were noted. It was found that staining protocols in which buffered saline was used as the diluent, instead of deionized water, decreased staining affinity, and thus, higher concentrations of methylene blue stain and prolonged staining times (30–60 min) were necessary in order to achieve ACF visualization. Whole mounts stained with this method resulted in ACF that appeared muted and were difficult to distinguish from the normal mucosa. The methylene blue stain in unbuffered deionized water required lower concentrations of methylene blue stain and a shorter incubation period; this approach resulted in vibrantly stained ACF that were discernable from the normal mucosa (Figure 2a–h). Vertical agitation of slides in methylene blue stain was required to achieve uniform staining. Optimal staining was observed when whole mount slide racks containing evenly spaced slides were fully immersed and withdrawn from freshly prepared methylene blue stain, 20 times at 1-s intervals and then allowed to sit undisturbed in methylene blue for 3 min. Attempts were made to mimic manual agitation using various mechanical devices, e.g., magnetic stir bar in a staining dish, an orbital shaker, etc. However, all of the mechanical devices evaluated proved inferior to the manual staining technique.
4.4 Image Acquisition
4.5 High Iron Diamine–Alcian Blue Stain
4.6 Image Analysis
The low degree of contrast between ACF and surrounding normal mucosa in methylene blue-stained whole mounts presented a significant challenge for automated image analysis. The authors are unaware of any software algorithm to date that consistently and reliably identifies ACF in methylene blue-stained colon whole mounts. Though this technological advance would likely shorten analysis time and improve reproducibility, the lack of such an approach necessitated direct user interaction. As opposed to the constraints placed on users of Image-Pro Plus® software, Adobe Photoshop® enabled ACF to easily be circumscribed using a non-destructive layer and improved work flow control. Use of Photoshop® was also key to developing overlay capability and studying the staining congruence and divergence of histological features from a single ACF. Multiple renderings of each ACF image were used to facilitate accurate selection of crypts prior to analysis. While many crypts are easily identified, there are instances where slight invaginations can masquerade as crypts. Use of surface plot enhanced images enabled greater definition of the two-dimensional image, which allowed the observer to discard false-positives prior to analysis (Figure 3).
4.7 Whole Mount Processing and Paraffin-Embedding
Obtaining flat tissue sections for embedding and microtomy was complicated by the tendency for the thin tissue to curl. The approach that we adopted to overcome this problem involved direct processing of the whole mounts while attached to glass slides, providing for adequate reagent transfer into the tissue and keeping it flat. An abbreviated processing schedule was used to decrease the amount of dehydration dependent shrinkage artifact. Tissue processed in this manner remained attached to the glass slide through all processing stages including paraffin infiltration. Attempts to use disposable plastic base molds proved ineffective while stainless steel base molds produced consistently flat paraffin blocks.
4.8 Microtomy and Staining
4.9 Morphometric Analysis of ACF
Incidence, total, and average number of ACF (non-MDF and MDF) by colonic region
9.6 ± 10.6
38.1 ± 14.7
26.1 ± 12.1
0.1 ± 0.3
0.9 ± 2.0
1.1 ± 1.5
Morphometric measurements for which aberrant crypts were evaluated
Description with units
Total number of crypts per ACF
6.7 ± 5.4
Area of each ACF in square microns (μm2 × 104)
2.5 ± 1.5
ACF epithelial area (μm2 × 104) (ACFAREA–CSUMAREA)
2.3 ± 1.3
Sum of crypt area (μm2 × 103)
2.2 ± 2.5
Average crypt area in square microns (μm2 × 102)
3.4 ± 1.7
ACF average diameter in microns (μm × 102)
1.7 ± 0.5
Crypt maximum diameter in microns (μm × 10)
3.0 ± 1.0
Crypt average size length in microns (μm × 10)
2.9 ± 1.1
ACF perimeter in microns (μm × 102)
5.6 ± 1.6
Average crypt perimeter in microns (μm × 10)
6.8 ± 2.7
ACF roundness (1 = round, higher numbers indicate polygonal shape)
1.1 ± 0.1
Average crypt roundness (1 = round, high numbers indicate polygonal shape)
2.2 ± 0.6
Crypt roundness category: 0–4
1.7 ± 0.7
0.5 ± 0.2
ACF integrated optical density (area × density) (μm2 × 104)
1.2 ± 0.9
Percent sulfomucin area (HID stain only) (%)
55 ± 32
Percent sialomucin area (AB stain only) (%)
16 ± 20
Percent unstained area (lacks sulfo- and sialomucin HID-AB stain; high numbers, e.g., ≥85% probably indicate MDF) (%)
30 ± 26
Mucin-depleted foci (≥85% PUAREA)
Morphometric measurements of ACF with crypts ≥5 that differentiate non-MDF and MDF
Non-MDF (n = 819)
32,207 ± 522
15,134 ± 336
195 ± 2
650 ± 5
MDF (n = 28)
25,820 ± 1429
10,995 ± 839
176 ± 5
593 ± 16
4.10 β-catenin Immunohistochemistry
4.11 Technical Effort
Estimated procedure and training time
Procedure time (min)a
Training time (min)b
Colon excision and cleaning
Whole mount preparation
ACF identification and analysis
Methylene blue stainingc
ACF image extraction
Methylene blue image analysis
MDF identification and analysis
Image layer alignment
MDF image extraction
HID-AB image analysis
The approaches detailed in this paper are currently being used to qualitatively and quantitatively compare carcinogen-induced premalignant colon pathologies based on morphological and biochemical characteristics of whole mount preparations. These methods, involving staining with methylene blue and HID-AB, followed by analysis of the same lesions using light microscopy and traditional histochemical and immunohistochemical techniques, should provide the following opportunities: (1) enhancement of mechanistic investigation into ACF molecular pathology and establishment of premalignant endpoints predictive of subsequent development of colon carcinomas; (2) the development of more robust screening approaches for preventive agents and dietary factors that impact colon carcinogenesis; and (3) improvement of the design of intervention studies in animal models, especially where changes in mucin metabolism occur during the disease process, such as inflammatory bowel diseases.
aberrant crypt foci
gut-associated lymphoid tissue
hematoxylin and eosin
high iron diamine–alcian blue
This work was supported by PHS grant U54-CA116847 from the National Cancer Institute. The authors would like to thank Jennifer Price, Joy Hester, Elizabeth Neil, and Jennifer Sells for their excellent technical assistance.
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