Diagnosis of Hirschsprung's Disease by Immunostaining Rectal Suction Biopsies for Calretinin, S100 Protein and Protein Gene Product 9.5

This method can help answer questions in the diagnosis of Hirschsprung's disease. The method and functions of this technique, if you follow protocol, exhibits high sensitivity and the specificity rates which process at the same time. Demonstrating the procedure will be Mijing Fang, a technician from my laboratory.

Begin by fixing the rectal suction biopsies in five times the volume of the tissue in 4%paraformaldehyde for six hours at room temperature. Dehydrate the fixed tissues in a pathological tissue dehydrator for 11 hours and embed the processed rectal suction biopsy tissues in paraffin blocks. Trim the blocks on a microtome until a complete section plane of the tissue is exposed.

Then use the microtome to obtain a 0.4 micrometer sections of the embedded tissue. Place in 45 to 50 degree Celsius water to allow the sections to spread into a flat plan. Transfer the flattened sections onto glass slides and bake them in a 60 degree Celsius oven for three to five hours.

Place the deparaffinized slides into two 15-minute changes of xylene followed by their hydration in sequential five-minute ethanol immersions. Then rinse the slides in reverse osmosis purified water for an additional five minutes. For antigen retrieval, place the slides in a Coplin staining jar of retrieval solution and place the jar into a preheated bath of boiling water.

After 20 minutes, allow the jar to cool to room temperature for about 10 minutes and gently rinse the slides with PBS. Then use a hydrophobic marker to encircle each tissue section and place the slides into a wet box. To block any peroxidates, add two or three drops of 3%hydrogen peroxide solution onto each tissue and place the slides at 37 degrees Celsius for 20 minutes.

At the end of the incubation, rinse the slides with three five-minute PBS washes followed by blocking with 5%bovine serum albumin for 20 minutes at 37 degrees Celsius. Next, discard the excess bovine serum albumin and label the cells with 50 microliters of the primary antibody of interest at 4 degrees Celsius overnight. The next morning, rinse the slides with three three-minute washes in PBS and add 50 microliters of polymer enhancer to each slide.

After 20 minutes at 37 degrees Celsius, wash the slides three times in PBS and add 50 microliters of secondary antibody B to each tissue section. After 30 minutes at 37 degrees Celsius, rinse the sections with three five-minute washes in PBS and add a drop of freshly prepared DAB working solution to each slide. When the appropriate level of brown staining is detected by light microscopy, rinse the slides in PBS for five minutes and counter stain the nuclei with one drop of hematoxylin for one minute.

Remove the excess stain under a trickle of water for approximately 30 to 40 seconds followed by a 25 to 30 second wash in fresh PBS. Differentiate the tissues in 1%hydrochloric acid ethanol for 10 seconds followed by a one-minute PBS wash. Dehydrate the slides with five-minute immersions in the reverse order of hydration and expose the slides to two five-minute changes of xylene.

Then mount the cover slip using ultra-clean mounting and allow the slides to dry before their visualization on a light microscope. In this representative study of 318 rectal section biopsy patients, 99 patients were initially diagnosed by rectal section biopsy. Many ganglion cells which express calretinin were observed in normal tissue biopsies.

However, no ganglion cells were detected in any area of the tissue from the Hirschsprung's disease segments. S-100 and PGP9.5 immunostaining revealed hypertrophied nerve trunks in the submucosa of the diseased tissue with only granular staining of some small nerve twigs observed in the normally innervated intestine. While attempting this procedure, it is important to remember to pay attention to details like size of rectal suction biopsies and thickness of sections.

After it is defragmented, this technique will pave the way for researches in the field of pediatric surgery to support a diagnosis of Hirschsprung's disease.