Using Unfixed, Frozen Tissues to Study Natural Mucin Distribution

The overall goal of the following experiment is to preserve the natural distribution of glycosylation, of secreted mucus in tissue samples. This is achieved by first embedding the tissues in optimal cutting temperature, medium or OCT to minimize the tissue processing, and then to allow detection of glycan structures, mucin and mucus. The sections are incubated with lectins antibodies and histochemical stains.

Lectin binding can be further challenged by competitive inhibition or enzymatic cleavage of glycan epitopes to confirm lectin binding specificity. Ultimately, the preservation of mucus and frozen tissue samples can be compared to the preservation of mucus in paraffin embedded samples by mystic chemical analysis. The main advantages of using this technique over existing methods such as embedding tissue in paren and eh fixing with car noise solution are that this method does not require the use of specialized fixatives and allows the utilization of frozen tissues that may already be existing in the lab.

To begin, allow the snap frozen tissue to warm up to minus 20 degrees Celsius by placing it in a cryo microtome chamber. Meanwhile, create a freezing bath by adding two methyl butane to a shallow styrofoam box and then adding dry ice to the box. Next, add just enough OCT to cover the bottom of a peel away freezing mold, and then place the tissue in the mold.

Make sure that the tissue rests on the bottom of the mold in the desired orientation. Cover the tissue with more OCT and then place the mold in the freezing bath. The OCT compound will turn white as the tissue freezes Once frozen, peel the mold off the frozen block and place the block in a marked freezer bag.

Store the frozen blocks at minus 80 degrees Celsius until use before sectioning the tissue. Place the frozen blocks of interest in the cryo microtome chamber for about 30 minutes to allow them to reach minus 20 degrees Celsius. When the blocks of warmth cut a three to five micrometer thick section and then place a positively charged glass slide on top of the section.

The tissue will adhere to the slide after air drying the tissues for 30 to 60 minutes. Fix the slides with 10%buffered formula in for 30 minutes at room temperature, and then wash the slides three times in PB PBS by 10 quick dips in 250 milliliters of buffer to stain tissues with aum. Blue first, rinse the slides in water and then incubate in 3%acetic acid for three minutes.

Then incubate the tissues with AUM blue solution After 30 minutes, wash the slides in running, tap water for 10 minutes and then rinse in DI water. Counter stain the nuclei with nuclear fast red for five minutes, and then wash the slides three times in DI water. Now dehydrate and clear the slides by incubating for one minute in 95%ethanol, followed by three quick changes in 100%ethanol, and then three changes in citrus resolve for two minutes each.

Finally mount a cover slip on the slide with a resonance medium such as cyto seal 60 to stain tissues with periodic acid shift. First, rinse the slides in water and then incubate with freshly prepared 1%periodic acid for five minutes. Now, wash the slides three times in di water.

Dip the slides once in Milli Q water and then stain the tissues with shiff reagent. After 15 minutes, wash the slides in running, tap water for another 10 minutes and then dip once in di water. Counterstain the nuclei with sgio metin for 30 seconds, and then wash the slides three times in DI water.

Finally incubate the slides for 30 seconds. In Scot's tap water bluing reagent wash three more times in DI water, and then after dehydrating the tissues as just shown for calcium blue staining. Mount the slides with a cover slip for fluorescence detection of glycan epitopes using lectins.

First, block the slide with BSA in PBS for 10 to 30 minutes. Then block the endogenous biotin by incubating the slide for 15 minutes with avadon, followed by a PBS wash, and then a 15 minute incubation with 0.01%biotin after another PBS wash. Place the slide in a staining box layer a freshly prepared mixture of the desired lectins on top of each tissue section, and then gently cover the lectin mixture with parfum.

Now incubate the slide in the dark after one hour. Gently remove the parfum and wash the slide three times with PBS. Then layer the slide with strep avid in conjugated SCI five and incubate it in the dark once again after 30 minutes, wash the slides three times with PBS and then counterstain the nuclei with dappy.

Finally, mount the slide with a cover slip using an aqueous medium such as vector mount mounting media for the sase enzyme control. Begin by adding water to the bottom of an empty tip box to form a humid chamber. Place the negative control slides face up on the top tray of the tip box.

Layer 150 to 200 microliters of a US solution onto the tissue sections, and then cover each slide with a cover slip, avoiding air bubble formation. Close the box lid and incubate the box at 37 degrees Celsius after two and a half hours. Wash the slides three times in PBS at room temperature to remove any free slic acids.

Then incubate the slides with SNA for an hour at room temperature for the competitive inhibition. Control aliquot 200 microliters of the lectin mixture to two einor vials. Then add 200 millimolar of the Jacqueline inhibitor Meli bios to one of the vials, and add the SWGA inhibitor chitin hydrolysate at a one to 10 dilution to the other vial.

Then incubate the slides with the inhibitor containing mixtures for one hour at room temperature. In this first figure tissue, sections of cumin mouse or chicken colon specimens frozen in OCT or embedded in paraffin were stained with periodic acid shiff in pink or calcium blue in blue. A comparison between tissue samples embedded in paraffin to frozen tissues embedded in OCT Cryoprotectant media revealed striking differences in the preservation and quality of staining for mucin glycoproteins in the frozen tissues.

In addition to mucin in goblet cells, secreted mucus was also visible as indicated by the arrows in the paraffin embedded tissues. The mucus staining was confined to goblet cells. In this figure, the substantial loss of mucin and tissues incubated in citrus resolve can be observed.

Serial sections of frozen chicken ileum specimens were fixed in 10%buffered formin and kept hydrated, dehydrated in ethanol by sequential incubation in 70%90%and 100%ethanol for 20 minutes each, or dehydrated in ethanol and then cleared with citrus all for one hour. Ethanol dehydration alone and ethanol dehydration plus citrus all clearing improved tissue morphology. Further ethanol dehydration alone had no significant effect on blue staining.

In contrast, citrus all incubation reduced and confined blue staining to goblet cells in a pattern that was similar to that observed for the paraffin embedded tissues seen in the previous figure. The boxes indicate the areas of higher magnification in the allium blue stain tissue sections. In this next series of images, chicken ileum specimens frozen in OCT or embedded in paraffin and then probed with Jacqueline in blue, SWG in green and SNA in red are shown in frozen tissues.

Jacqueline binding to olink glycans revealed structures that appeared to be oozing from goblet cells into the lumen as indicated by the arrows in the OCT frozen tissue section. In contrast, Jacqueline binding to paraffin embedded tissues was confined to goblet cells and to the villi brush border indicated by these arrows. SWGA staining of beta one four GL partially co localizes with the binding of the Jacqueline lectin in both frozen and in paraffin embedded tissues as indicated by the arrows.

In contrast, SNA lectin in red and indicated by the arrow heads binds intracellularly to alpha two six linked sialic acids and does not co localize with Jacqueline, which is blue and is indicated with the dotted arrows In this figure. Slic acid epitopes were cleaved from chicken ileum specimens by digesting the tissue section with a US or were left cleaved by incubation and sodium acetate buffer as seen here. The a US treatment abolished the staining with biotinylated SNA that is observed in the untreated tissue confirming SNA binding specificity to sialic acids.

Lectin specificity can also be confirmed by adding a competitive inhibitor such as mely bios for Jacqueline staining or chitin hydrolysate for SWGA staining chicken ileum specimens, once again, were incubated with a Jacqueline and SWGA mixture. This time also in the presence of specific lectin inhibitors, mely bios or chitin hydrolysate Jacqueline staining was inhibited by mely bios, but not by chitin hydrolysate. Conversely, SWGA staining was inhibited by chitin hydrolysate, but not mely bios.

This final series of images demonstrate that snap frozen tissue samples, which are routinely obtained in the clinic and in research laboratories can be further embedded in OCT and used to study glycans on mucin, glycoproteins, glycolipids, and glycans. These colorectal cancer biopsies from villous carcinoma and mucus carcinoma tissues were snap frozen in liquid nitrogen embedded in OCT and successfully stained for the various indicated mucin and glycan epitopes. After watching this video, you should have a good understanding of how to embed tissues in OCT and how to analyze mucus and tissue glycosylation using immunochemical methods.