Deficient Pms2, ERCC1, Ku86, CcOI in Field Defects During Progression to Colon Cancer

The overall goal of this procedure is to determine the particular alterations in DNA repair protein, expression, or expression of proteins related to apoptosis that are frequent in areas surrounding colon cancers and thus likely to be central to progression to colon cancer. This is accomplished by first collecting tissue samples in formalin jars, either from colonic mucosa, from normal colonic mucosa during colonoscopies as controlled tissues or colonic mucosa adjacent to colon cancers from colon resections as experimental tissues after formal and immersion for four hours. These tissues are transferred 70%alcohol overnight, followed by embedding in paraffin blocks.

The second step of the procedure is to cut tissue sections from the blocks and transfer them onto microscope slides. The third step of the procedure is to immunostain the tissue sections for possible proteins of interest using a sequenza rack. The final step of the procedure is to view the tissue sections in a photo microscope.

Ultimately, results can be obtained that show high levels of expression of one or more proteins in colonic mucosa that has no local neoplasia and reduced expression of these proteins in field defects surrounding colon cancers indicating the importance of their reduction in progression to colon cancer. For example, this image shows brown staining indicating high levels of expression of E rcc, one in colonic mucosa without colonic neoplasia. This next image shows brown staining indicating high level of expression of PMs, two in colonic mucosa of a patient without colonic neoplasia, while an area of colonic mucosa near a colon cancer shows one crypt with high PMs, two expression and some with low expression, and another area near a colon cancer with absent expression of PS two.

Hi, I'm Hoy Wynn from the laboratory of Carol Bernstein in the Department of Cell Biology and Anatomy in the College of Medicine at the University of Arizona. I'm Kristi Luo. I'm Alex fta.

I'm Nadia Suna and we're all from the Bernstein Lab. Today we'll show you tissue sections taken during a colonoscopy and a procedure for cutting sections from these tissue samples. We will then demonstrate the procedures for immunostaining these tissue sections.

We will also show you how we examine our tissue sections with our microscope. The tissue sections are examined for the expression of proteins which may be relevant to the progression to colon cancer. We use these procedures to find that deficiencies in 2D NA repair proteins.

ERC one and PMMS two appear to be among the first steps in initiation to a field defect that can lead to a colon cancer. We also use these procedures to eliminate other proteins of likely importance in early steps in progression to colon cancer such as DNA repair protein CO 86 and apoptosis related protein CCO one, So let's get started. Biopsies of normal colonic mucosal tissues are obtained from patients during a screening colonoscopy and placed in jars of formin.

After four hours, the formin is replaced with 70%alcohol. Similar samples of tissue are obtained from areas of colon resections one centimeter and 10 centimeters away from colon cancers are adenomas with advanced neoplasia. The tissue samples are sent to a histology laboratory for processing into paraffin blocks prior to sectioning.

The paraffin blocks are chilled in a refrigerator to make them easier to cut by a microtone cut tissue sections of four microns in thickness from the tissue blocks and float them on water for comparing the expression of two proteins. For instance, ERC one and pmms two pick up alternate tissue sections onto two slides until there were three tissue sections on each of two slides. For example, sections one, three and five can be placed on one slide labeled ERCC one and sections two, four and six can be placed on one slide labeled PMS two.

The procedure for immuno staining. The previously prepared tissue sections should be carried out under a chemical hood with negative air pressure so that the experimenter is not exposed to the vapors. The protocol we use is a fairly standard one, however, to get results similar to ours.

When you use this protocol in your lab, adjustments may be needed to take into account variables such as water quality, microwave power, and concentration of antibodies. In this video, we will demonstrate PMs two immunohistochemistry first de parize the slides by placing them in three changes of xylene for three minutes each, followed by three changes of two minutes each in 100%ethanol, followed by two changes of two minutes each in 95%ethanol. Finally wash the slides in two changes of distilled water.

Next, place the slides in a buffer solution and bring to a boil in a microwave oven. This is followed by 10 minutes on the medium setting. This step, which is called heat induced epitope retrieval, breaks the cross links formed in the tissue sample during formal in fixation and enables the antibody to recognize the protein of interest after cooling on ice for 20 minutes.

The slides are washed in PBS and distilled water for three minutes each. The slides are then placed in a solution of 3%hydrogen peroxide for 20 minutes. The purpose of this step is to block endogenous peroxidase activity in the tissue.

Furthermore, by blocking this activity, we reduce the non-specific background staining that may be caused by the antibody after 20 minutes in the endogenous peroxidase block, the slides are washed in distilled water and then PBS for three minutes each. For immuno staining, place the slides into flat narrow staining racks called sequina and rinse with PBS. The slides being stained for PS two or ERCC one are then exposed for 10 minutes to an added treatment of three drops of a proprietary solution called sniper, which acts to reduce non-specific staining of background proteins.

After rinsing with TBST buffer, the slides are incubated with the antibody to PS two after two hours, rinse the slides three times with buffer and then add 100 microliters of biotinylated secondary antibody. Add a one to 100 dilution incubate for 30 minutes at room temperature after 30 minutes, rinse the slides three times with TBST buffer. Add three drops of vector stain A B, C reagent to each slide and incubated room temperature for another 30 minutes after the incubation.

Wash once with TBST and transfer the slides from the sequina into a slide rack in PBS. The slides are later immersed in dab substrate for up to five minutes with watching to ensure that the brown staining does not get too dark. This is followed by counter staining with a dilute solution of hematin For 10 seconds, rinse slides thoroughly and tap water followed by deionized water, dehydrate the slides two times for two minutes each in 95%ethanol two times for two minutes each in 100%ethanol and two times for two minutes each in xylene.

Finally, add a couple drops of mounting medium to each slide and apply a cover slip. Now, Hein DW will examine a stained tissue section under a modic photo microscope for expression of ERCC one in Crips of the colonic mucosa. We'll look first at a section of colon tissue under a moic photo microscope with a 20 x objective lens.

The yellow arrow is pointing to a colon crypt. There are roughly 200 colon crypt cells in this crit. These are the S stroma cells.

This is a crip lumen and this is the colon lumen in which feces passes through. There are three different types of colon crypt cells. Goblet cells are shaped somewhat like a wine glass and their nuclei are at the outer edge of the crypt shown here and highlighted in red.

They also have a ballooning out section filled with white mucin granules in the part of the cell on the inside of the crypt shown here. The other two types of cells look somewhat alike in our stain sections. They are the enterocytes and enteroendocrine cells and their nuclei are also at the outer edge of the crypt at the base of the cells indicated here by the red circles.

This particular section was stained for ER CC one. The brown stain shows the location of EER C one. Almost all of the nuclei in all Crips in this section show normal high level of expression of ER CC one.

The blue stain is hemat toin, which stains DNA in nuclei as shown here. The nuclei in the crypt have more brown than blue because of the high expression of ERCC one. Now we will navigate through the tissue with the 10 x objective lens.

Notice how most of the colon cribs are showing high levels of EER CCC one expression. Here we have an area with a few Crips showing lower ER CCC one expression indicated by the blue staining nuclei. As we approach the end of the tissue, we notice another area with lower ER CC one expression.

We'll now go back and look more closely at the Crips that were deficient for ER CC one. Looking with the 20 x objective lens, we can see the lower EER CC one express encrypt next to a higher EER CC one express encrypt. Notice how ERCC one protein is more heavily expressed in the cytoplasm of the deficient crypt indicating that the ER CC one is produced but unable to get into the nuclei.

Now with the 40 x objective lens, we can see more clearly that the ERCC one was unable to get into the nuclei of this deficient crypt compared to the highly expressing nuclei of the encrypt with little ER CC one in its cytoplasm, this tissue was taken as a biopsy from a patient with a previous colon cancer. Next, Nadia Una will examine a stained tissue section for expression of C cco one encrypts of colonic mucosa. We can go through a tissue section immuno stain for cytochrome C oxidase one or CCO one using a 40 x objective cco.

One is a cytoplasmic protein and is non expressed in the nuclei, which is why the nuclei have stained only blue CCO one is expressed in the cytoplasm shown with the brown staining. In this field of view, we can see two colon crips, one having positive expression of CCO one and one having a deficiency in CCO one expression panning through this tissue section. You'll notice that the majority of the colon crips have a positive expression of CCO one.

Now Hoy Quinn will show CCO one immuno stain Crips indicating the stem cell region of the crypt and its function. We are now looking at a section of tissue stain for cco. One reports indicate there are less than five stem cells at the base of the crypt given rise to the 2000 total cells of the crypt.

The stem cell region is shown in the red box indicated here. A stem cell with a mutation or an epimutation can take over the whole stem cell niche or half of the stem cell niche. Here we have Crips that are showing high normal expression of ccl, one indicating stem cells without a mutation for one.

Here we have an example of how a mutation has resulted in altered staining of a whole crypt stain. For CCL one, notice the lack of CCL one expression throughout the entire crypt, especially when compared to a normal staining crypt. Here we have an example of how a mutation has resulted in altered staining in half of a crypt.

Notice how this section of the crypt shows low levels of CCO one expression when compared to this section of the crypt, which shows high level of CCO one expression. Now, Nadia Suna will show CCO one immunostain Crips in cross-section and indicating that some Crips have cross-sections arising from mutant stem cells lacking cco. One Here is a tissue section immuno stain for CO one showing colon Crips cut crosswise.

We will observe this section using a 20 x objective. There are two pairs of fishing Crips showing positive expression of CO one and one fishering structure showing CCO O one deficiency in one crypt. This crypt shows deficiency of CCO O one expression in the whole structure where all of the crypt stem cells became CCO one deficient cells.

These two crypts show half conversions where approximately 50%of the stem cells were deficient in CCO one expression. These stem cells gave rise to the deficient half. Now, Alex FTA will examine a tissue section immuno stain for CCO one and identify elements of the tissue For the purpose of identifying some other structures in the tissue.

We will observe a section stain for CCO one with a 10 x objective lens indicated by the red box is a lymphoid nodule, the luminal edge ryt interstitial cells also called S stroma cells and the muscularis, which extends down the tissue. Now we will look at a slide showing tissue obtained from the normal appearing area of a resection when a patient had a segment of his colon removed. This area very near a colon cancer can be considered to have a field defect from which the cancer arose here.

Some Crips have higher levels and some have lower levels of expression. For ERCC one Christi Luau will show us how this tissue is evaluated. Here is a tiled image taken with a 10 x objective lens of a tissue section closely adjacent to a cancer and it is stained for ERCC one next to a cancer.

Many other crips are frequently deficient for ER CCC one. We will start to evaluate the level of ER C one expression using a 40 x objective lens and the Crips are given a score from zero to four depending on the expression level of ER CC one. Each tissue section on the low resolution image is then dotted with a color blue for high expression level four green for fairly high expression, level three yellow for reduced expression, level two orange for a very low expression level one and zero for absent expression.

Level zero. Here is the first cry we will be scoring. As you can see, the nuclei along this edge are almost completely deficient.

On the other edge. There is a small amount of expression and we label this crypt level one for very low staining and dotted orange. This crypt has a fairly high amount of staining in almost all of its nuclei getting a green dot for level three.

By comparison, all of the nuclei at the lumen have very high level four staining. This crypt has low expression of ERC one along this edge, so we. it yellow for level two.

This crypt exhibits both high expression on one edge and fairly high expression on this edge, and we place a green dot for overall level three expression. This crypt has reduced expression of ERC one and is marked level two with a yellow dot. This crypt has high level four expression and we give it a blue dot.

Overall, this appears to have reduced expression level two, getting a yellow dot. We consider crips level zero, one and two red, orange and yellow to have reduced or absent expression, whereas level three and four, green and blue are considered to have relatively high expression. This tissue section had 44 crips with low expression and 23 crips with relatively high expression.

Thus, 67%of Crips in this tissue section near a cancer had reduced or absent expression of ERCC one Tissue sections. Cut one after the other from a tissue block with alternate cuts placed on separate slides allow us to look at the same colonic Crips on two slides. These tissue sections were obtained from tissue very near a colon cancer.

We stained one slide with antibody to ERC one and the other slide with antibiotic to PMs two. These tissue sections are shown by Christi Luau. Here we have one of two sequential cut tissue sections near colon cancer amino stained for EER CCC one on a 10 x objective lens.

You can notice ER CCC one is highly stained near the lumen, although most of the crips are not stained. You can also notice the same round staining in the interstitial cells at a higher magnification. We see this crypt has high expression of ERCC one.

Here we have a second sequential cut tissue section near colon cancer immuno stain for PS two. As we move through this tissue section, the crypt which has stained for ER CCC one, has also stained for PMs two. At a higher magnification, we see high expression of pmms two in the same crypt.

Here, both crypts are paired. You could notice the high expression of ERC one here. Similarly, the same crypt stained for PMs two also has high expression.

These neighboring crips do not exhibit high expression shown here and here adjacent Crips deemed for PMs two also do not show high expression here and here. We also use immunohistochemistry to determine the frequency of Crips deficient for KU 86 and CCO one. On this slide, poin will go through an entire tissue section obtained near a colon cancer and immuno stain for KU 86.

We see that a few Crips are deficient for KU 86 in this cancer associated tissue section. We also use immunohistochemistry to determine the frequency of Crips deficient for K Kuku 86 and CCL one ne a colon cancer. On this slide, we go through part of a tissue section immuno stain for KU 86 using a 10 x objective lens.

As we navigate through the tissue, we see that most crypts show high level of expression of KU 86. However, as will be indicated by the red boxes, there are a few Crips that are deficient in KU 86. On this slide, Alex FTA will go through an entire tissue section obtained neuro colon cancer and immuno stain for CCO one.

We are now looking at a tissue section taken one centimeter away from a colon cancer immuno stain for CCO one with a 10 x objective lens. We can observe a crypt with high expression of CCO one as well as a cluster of crypts with low levels of expression of cco. O one here is efficient encrypt crypt.

This portion has high level of expression, whereas this portion has low level of expression. As I pan through this tissue section, we can notice that most of the Crips have a high level of expression. I have previously tallied all of these Crips.

There's a total of 189 Crips and 45 of them are deficient in cco one that gives a frequency of about 23.8%deficient in this crypt. About half of the crypt is highly expressing NCC O one, whereas half of the crypt is deficient in CCC O one, we note that CCO one deficient Crips are not as frequent near colon cancers as PMMS two or ERCC one deficient Crips CCO one deficient Crips tend to have a frequency of 10 to 20%deficient, whereas ERCC one and PMs two tend to be around 70 to 90%deficient near colon cancer. We've just shown you how to cut an immuno stain tissue sections from colonic mucosa for proteins that may be important in progression to colon cancer.

We've also shown you how to evaluate these tissues for deficiencies in certain proteins that are in the Crips. When Doing this procedure, it's important to have three tissue sections per slide. Bubbles can occur when applying the media, carrying the antibody, preventing Crips or portions of Crips from standing adequately just because a bubble prevents the antibody from reacting with the tissue section.

Crips are usually 60 microns in diameter and tissue sections are four microns thick, so there could be as many as 15 tissue sections through the same crypt. It is usually possible to find the same crypt using the morphology of the crypt as a guide on multiple tissue sections placed on a slide. So that's it.

Thanks for watching and good luck with your experiments.