Techniques for Modeling Human Intestinal Cancer in Mice

October 22-26, 2000

Presented by

The Jackson Laboratory

Faculty: Catherine Booth, William F. Dove, Jeffrey I. Gordon, Richard B.
Halberg, Julie Ann O\'Shea, Thaddeus S. Stappenbeck, and Robert H.
Whitehead

Edited by: W. F. Dove, R. B. Halberg, L. Clipson,

and I. L. Riegel

The McArdle Laboratory for Cancer Research

University of Wisconsin-Madison

Send any corrections or additions to dove@oncology.wisc.edu

4.  ***In Vivo* Crypt Analysis**

# 

# Crypt Microcolony Assay

######## Catherine Booth

######## Paterson Institute

This is an assay that allows the determination of the number of
surviving crypts (and therefore clonogenic cells) that may be present in
the small and large intestine following cytotoxic insult. It can be used
to gain an understanding of the effects of various factors on the
clonogenic cells in the intestine and can provide basic radiobiological
information. It can also be used to test the efficacy of various agents
that may protect the intestine from the effects of cytotoxic agents
(mucositis prevention).

Adult animals (mice) are irradiated using either Gamma or X-ray sources
at a range of doses from 8--16 Gy, or treated with a cytotoxic drug at a
range of predetermined doses and then sacrificed 4 days later. In the
case of radiation the doses may be administered as either whole or
partial body radiation. The ideal group size is 6 mice, which are then
exposed to one of the doses of irradiation or used as a control group
(untreated animals).

At the time of sacrificing, the small intestine is removed and flushed
out with saline with a 21G needle and syringe, or alternatively the
luminal contents are removed by scraping along the exterior of the
intestine pushing the contents to the end. The tissue is then fixed in
Carnoy\'s fixative for 30 min and then changed to 70%-ready for tissue
processing.

In order to obtain absolute cross-sections of the intestine, it is
important that the tissue is orientated in the correct manner. This can
be achieved by cutting the intestine into 10-12 pieces, and then
collecting these pieces together to form a bundle inside a loop of
surgical micropore tape. The tape can then be tightened to pull the
bundle of tissue together so that it has the appearance of a compact,
tightly held pile of logs. The uneven edges of the bundle can then be
trimmed by using a scalpel blade so that the bundles become a suitable
size for embedding and sectioning. Once bundled, the tissue should be
placed into a labeled histology tissue cassette and placed in 70%
ethanol. If tissue is processed correctly, it is possible to place all 6
samples from an experiment group into one cassette. Care should be taken
to keep the tissue moist while preparing each bundle, to deal with one
intestine at a time, and once bundled place the tissue into the tissue
cassette and place the cassette in a petri dish containing 70% ethanol
to prevent drying out until all 6 samples have been processed. Once
embedded, the tissue should be sectioned (3--5 micron sections are best)
and stained with hematoxylin and eosin. From each bundle, 10 good
cross-sections of intestine per mouse can be obtained.

The determination of the number of surviving crypts present per mouse is
determined in the following manner:

1.  From each bundle, 10 circumferences must be counted; these
    circumferences must be intact and not contain Peyer\'s patches.

2.  Determine the number of surviving crypts that are present in each
    circumference. A surviving crypt is one that has 10 or more
    intensely stained cells present (in addition to any Paneth cells).

> It should be noted that not all crypts will be embedded straight, so
> that when sectioned they may appear as 2 small crypts superimposed or
> adjacent to each other. Crypts such as these, therefore, should be
> counted only as a single crypt.
>
> Also, crypts may not be perfectly sliced along the central lumen when
> sectioned. Such crypts may appear as a ball of cells without a lumen
> and can still be scored if they contain the requisite number of cells.

The size of the surviving crypts present will vary, depending upon the
type of treatment to which they have been exposed. This will influence
the likelihood of observing a surviving crypt in cross-section. This
error can be reduced by applying a size correction factor. To do this,
the widths of the surviving crypts must to be determined. Generally, 15
widths measurements are taken from each circumference so that 60 widths
per group of mice are measured. Again, there are criteria for the crypts
which should be measured. They should:

> i\. Satisfy the criteria of a surviving crypt.

ii. Have Paneth cells at the bottom of the crypt.

iii. Have a clear lumen through the center of the crypt.

iv. The width of the crypt should be measured at the widest point.

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3.  The size correction can then be included in the equation below,
    giving the number of surviving crypts present that have been size
    corrected.

> Corrected crypts= Mean no. surviving crypts per X [Mean crypt width
> for an untreated group]{.underline}
>
> treatment group Mean crypt width for treated group
>
> If the effects of various protective agents are to be administered,
> then they are usually added as a pre- or post-irradiation treatment,
> with the animals still being sacrificed 4 days post insult.
>
> **Unirradiated, 2.5x**
>
> ![](media/image1.png)
>
> **Unirradiated, 10x**
>
> ![](media/image2.png)
>
> **Irradiated, 10 Gy, 10x**
>
> ![](media/image3.png)
>
> **Irradiated, 10 Gy, 10x**
>
> ![](media/image4.png)
>
> **Irradiated, 12 Gy, 10x**
>
> ![](media/image5.png)
>
> **Irradiated, 14 Gy, 10x**
>
> ![](media/image6.png)
>
> **Irradiated, 16 Gy, 10x**

![](media/image7.png)

**Bromodeoxyuridine (BrdU) Staining**

######## Catherine Booth

######## Paterson Institute

Studies of proliferation in the mucosal tissues are of great importance
in that many diseases associated with the intestine arise owing to
alterations in the rates of cell proliferation within the intestine.
Various approaches can be used to study the cell kinetics of the small
and large bowel, such as ^3^HTdR or BrdU labeling, or Ki67 and PCNA
staining. The latter is not to be recommended, since results can be
highly variable and the protein is present through much of the cell
cycle. BrdU and thymidine labeling generally tend to be the most
reproducible and consistent means of assessment.

BrdU is a derivative of deoxyuridine that is specifically incorporated
into the DNA of proliferating cells during the S phase of the cell cycle
(S.Chwalinski 1988). BrdU labeling is a method that allows the study of
cell kinetics via visualization and analysis of the spatial distribution
of DNA synthesizing cells in the colon and small intestine, and the
frequency at which these cells occur. It is a means of examining the
labeling index (LI%) at each cell position, the data that can be
analyzed with the program New Crypts, and it is a suitable alternative
to thymidine labeling and produces similar results (Kellet et al. 1992).

**The Boric Acid Buffer Method**

1\. Dewax slides in xylene overnight.

2\. Air-dry and mark around tissue with a PAP (hydrophobic) pen.

3\. Wash 3 times in absolute alcohol for 5 min each.

4\. Block endogenous peroxidase activity with 1% hydrogen peroxide for
30 min (diluted in methanol).

5\. Place slides in 1N HCl at 60ºC for 8 min.

6\. Neutralize slides in boric acid buffer for 6 min.

7\. Wash in PBS for 10 min. Repeat this 3 times.

8\. Block with normal rabbit serum 1/20 for 30 min.

9\. Tap off serum and add primary antibody rat anti-BrdU 1:5 (Sera labs
Mas 250b) for 1 hr.

10\. Wash for 5 min in PBS, 3 times.

11\. Add peroxidase-labeled secondary antibody rabbit anti-IgG rat (Dako
P0162) 1:100 in 10% normal mouse serum for 1 hr.

12\. Wash for 5 min in PBS, 3 times.

13\. Immerse slides in DAB for 6 min.

14\. Rinse in distilled water.

15\. Place into 80% methanol for 10 min and then into thionin for 6 min.

16\. Dip slides 5 times in 2 changes of 95% alcohol and then place in
absolute alcohol for 1 min.

17\. Place in xylene for 10 min and then mount slides.

Boric acid buffer:

Stock A: 0.2 M orthorbic acid solution (BDH 1005845) (7.44 g in 600 ml
distilled water).

Stock B: 0.05 M sodium tetraborate (Borax) (BDH 30717) solution (9.5 g
in 500 ml distilled water).

To make buffer, add 550 ml of Stock A to 450 ml of Stock B and adjust
the pH to 8.4.

**MIB1 Monoclonal Antibody**

-   Dewax slides for approximately 30 min.

-   Rehydrate through a series of alcohols to water.

-   Microwave slides for 25 min on high power in citrate buffer (pH
    6.0); cool for 15 min.

-   Block endogenous peroxidase activity -- 0.3% H~2~O~2~ in PBS for 20
    min (3 ml in 300 ml).

-   Draw around the section with a PAP pen.

-   Block nonspecific binding with normal goat serum 1/10 for 30 min.

-   Primary Ab: mouse monoclonal (Coulter LTD) 1:50 with 10% normal goat
    serum for 1 hr at room temperature.

-   Wash in PBS for 2 x 5 min.

-   Secondary Ab: goat-anti-mouse Biotin 1/200 with 2% normal human
    serum for 30 min.

-   Wash in PBS for 2 x 5 min.

-   Vector ABC for 30 min.

-   **MAKE UP 30 MIN PRIOR TO USE**

-   Wash in PBS for 2 x 5 min

-   DAB for 5 min (200 μl of H~2~O~2~, 200 ml of PBS, and 1 vial of DAB)

-   Rinse in distilled water for 5 min

-   Counterstain with thionin