Staining Protocols for Human Pancreatic Islets

The overall goal of this procedure is to characterize human pancreatic eyelets using hematin and eosin staining and immunohistochemistry. This is accomplished by creating serial sections from a pancreas sample embedded in a paraffin block. The sections are then placed on slides in their original orientation.

A portion of the slides are stained with hematin and eoin while the remaining slides are stained by immunohistochemistry, the final step is to scan the stained slides and organize them by case. Ultimately, these images can be uploaded to an online pathology database where they can be viewed by other researchers in the field. The main advantage of this technique over existing methods is that slides are stained in series while maintaining their original orientation.

Demonstrating the procedure will be Linda Snyder, histo technologist, Emily Montgomery, biological Scientist, and Tiffany Hippo, laboratory technician in my laboratory. To begin set up the water bath and microtome following standard procedures for paraffin microtomy. Next label positively charged slides with the case number, pancreas region, and slide number.

Once everything is prepared, place the paraffin block in the microtome chuck with the cassette label on the left side. Following normal microtomy procedures, section the block until the tissue is uniformly exposed. Once the tissue is evenly encountered, produce a ribbon of serial sections about four microns thick.

Three to six sections are needed depending on the number of initial stains required. Next, separate the sections of the ribbon. Pick up each section in order and place it on a slide.

Record the section number with a pencil. If a section is unusable, skip that number and continue numbering the sections in exact order. Take care to keep the same orientation on the slide as found in the cassette with the slide label oriented to the left.

Next, place the slides in a slide rack and let them dry overnight. At room temperature, reseal the surface of the paraffin block with a thin layer of paraffin to preserve the tissue and archive it at room temperature or minus 20 degrees Celsius. Repeat this procedure for the next sample.

It is important to maintain the numbering for serial sections at each level as seen above of for h and e staining. Place the first slide from each block in a slide rack, and then place the rack in the first tray of the auto stainer. Select the standard HE program and start the staining process.

When the staining is finished, remove the slides from the auto stainer and place them in the hood for cover slipping. Place a cover slip on each slide using standard techniques. Lastly, label the slides with a printed label and allow them to dry thoroughly in the hood.

Begin by setting up the auto stainer and preparing all necessary reagents. Choose the Daco program for double stains. Input the numbers of slides and the treatment that each will receive.

Load all reagents into the auto stainer according to manufacturer’s recommendations. Next, prepare TBST and citrate, buffers, antibodies, and other reagents required for the procedure. De parize the slides by placing them in xylene for five minutes.

Do not allow the slides to dry out as this will result in excessive background and pore staining while the slides are depa. Pour citrate buffer into a steamer to preheat before use. Next, submit the depa slides to a series of ethanol washes.

Rinse the slides one last time in deionized water. Add the slides to the preheated citrate buffer and allow them to steam for 30 minutes. After this period, immediately transfer the slides to a room temperature water bath until they cool completely.

Once cooled, transfer the slides to the Dayco auto Stainer racks, taking care to maintain the proper sequence. Finally, run the double staining program that was loaded earlier when the double staining program has completed. Remove the slides from the auto stainer and let them dry for at least one hour before dehydrating.

To continue the dehydration, transfer them to 80%ethanol for one minute. Continue dehydrating the slides by soaking them in 95%ethanol and then 100%end. Lastly, dip and hold the slides in xylene for one minute.

Immediately mount the slides with cover slips using cyto seal To complete the procedure. Print labels that contain the case information, including the organ and block number, type of stain and date. Place a label on each slide to scan the stain slides.

Place them on a scanner tray and follow standard procedures. Archive the stain slides at room temperature and any remaining unstained slides at minus 20 degrees Celsius for future use. Finally, organize the stained images by donor and tissue type seen.

Here is an example of KI 67 plus insulin staining at both low and high levels of magnification. Images of the stained pancreatic sections are submitted to an online pathology database creating a virtual biobank. Using this database, investigators can rapidly access information needed for research and type one diabetes.

After watching this video, you should have a good understanding of how to prepare and stain high quality slides from the human pancreas.