Immunohistochemistry

Immunohistochemistry is a method used to localize a particular protein in a tissue. Detecting the protein's immunoreactivity with an antibody directed towards it does this. By determining the cell type expressing Fas and Fas Ligand in a tissue, one can postulate on the role of the Fas/FasL system in apoptosis of the tissue 8,13,15-17.

Reagents
1.Bovine Serum Albumin (BSA) – Sigma, St. Louis, MO
2.Saponin solution (0.1%) = 0.1 g saponin/100 ml PBS. For example, weigh 0.5 g of saponin and add to 500 ml of PBS. Keep stock at 4C for future use.
3.Triton (1%) – Add 5 ml of 100% Triton to 500 ml PBS. Place in warm water bath to dissolve. (0.5%) – Add 100 ml of 1% Triton to 100 ml PBS. Prepare in advance and store at room temperature for future use.
4.ABC Kit – Vectastain, Peroxidase Standard, PK-4000, Vector Laboratories, Burlingame, CA
5.DAB Kit – Peroxidase Substrate Kit, Vector Laboratories, Burlingame, CA
6.Harris Hematoxylin Solution (7.5 g/L), Sigma, St. Louis, MO
7.Whole Goat Serum – Organon Teknika Corp, West Chester, PA
8.Normal Horse Serum – Vector Laboratories, Burlingame, CA
9.FasL (N-20) – Rabbit polyclonal, Santa Cruz Biotechnology
10.Fas (B-10) – Mouse monoclonal, Santa Cruz Biotechnology
11.Biotinylated Anti-rabbit IgG made in goat, Vector Laboratories, Burlingame, CA
12.Biotinylated Anti-mouse IgG made in horse, Vector Laboratories, Burlingame, CA
Buffers and Medias
1.Phosphate Buffered Saline
2.Dilution Buffer (1%) = 0.1 g BSA/10 ml saponin solution. For example, weigh 0.05 g BSA and add to 5 ml of saponin solution. Prepare on the day of experiment.
Day 1-Afternoon

Chemical Hood Preparation:
Label glass immunohistochemistry trays: Xylene (4), 100% EtOH (3), 70% EtOH, 50% EtOH, MetOH/ H202, DD H20, PBS, 1%Triton, 0.5% Triton, and Hematoxylin. Make sure there is enough liquid in each glass tray to cover the slides entirely (about 200 ml). Xylene, hematoxilin, and Triton may be reused for multiple IHC’s, but everything else should be replaced with each use.

Antibody Solution Preparation: Prepare dilution buffer: For each slide, you will use approximately 100 (l of each antibody solution. Add the antibodies to the D.B. (Dilution Buffer).

Slide preparation:
Organize and record slides to be used. Place slides on slide warmer for 10-15 min. at 60°C

Deparaffinization
1.Dip rack of slides into first xylene tray. After 3 min, move rack to the second, third, and a fourth xylene tray for 3 min each.
2.Perform 3 changes of 100% EtOH for 3 min each. Place rack in methanol/ H202 solution for 30 min. Prepare by adding 30 ml of 30%H202 plus 200 ml 100% methanol to tray. This step quenches the endogenous peroxides.
3.After 30 min, return the rack to 100% EtOH for 3 min.
4.Place tray in sink and slowly introduce ddH20 into the tray of 100% ethanol until it is diluted to 0% ethanol. This takes about 5 min.
5.Wash in PBS for 5 min.
Blocking
1.Apply 100ml of the appropriate suppression serum solution according to the origin of the 2° antibody. (E.g. For anti-mouse antibody made in horse, add horse serum). In order to cover the tissue completely, turn the pipette horizontally, and drag the liquid across with the tip, being careful not to touch the tissue.
2.Place the slide in the incubation chamber and repeat with the remaining slides.
3.Cover the incubation chamber with a foil-wrapped lid, and incubate at room temperature for 20 min.
Primary Antibody
After incubation, use a Kimwipe to remove excess serum.
As with the suppression serum, add 100 ml of the 1° antibody solution to each slide.
Cover the incubation chamber with the lid and incubate overnight in 4C. Make sure that the tray is level in the refrigerator.
Day 2-Morning
Secondary Antibody
1.Return slides to rack and wash in PBS for 3 min. Repeat once.
2.Quickly dunk rack in 1% Triton 1-2 times.
3.Wash in PBS for 5 min with agitation (gently lift rack up and down). Repeat this for 2 changes.
4.As done previously, wipe the slides free of excess PBS and apply 100 ml of the appropriate secondary antibody solution.
5.Cover tray and incubate at room temperature for 1 hour.
6.30 min prior to use, prepare the ABC kit: Add 5ml PBS + 1 drop A + 1 drop B. Mix thoroughly and set aside until use.
7.After an hour of incubation in secondary antibody, wash the slides in PBS - 3 changes, 3 min. each.
8.Again remove the excess PBS and add to each slide 100 ml of the ABC previously prepared.
9.Cover tray and incubate at room temperature for 30 min.
10.Wash in PBS - 3 changes, 3 min. each.
Detection
1.Prepare DAB from kit. Always wear gloves when working with DAB because it is a carcinogen. Only make DAB when you are ready to use it because it is light sensitive. Make sure to mix between each addition. 2.5ml PBS + 1 drop buffer + 2 drop DAB + 1 drop H202
2.Place rack of slides into 0.5% Triton solution for 30 sec.
3.Using a Kimwipe, remove excess Triton from slide.
4.Apply 100 ml of DAB to each slide and watch for development. This will take from 3-7 min, depending on the type of tissue.
5.Once a slide is adequately developed, place it in ddH20.
6.When all the slides have been returned to the rack of ddH20, place the tray in the sink and rinse continuously with ddH20 for about 1-2 min.
Counterstaining and Mounting
1.Place rack in Hematoxylin for 10 seconds then place in tap water.
2.Rinse continuously with warm tap water for 2-3 min, until the water is no longer colored.
3.Begin a series of Ethanol gradients: 50% EtOH, 70% EtOH, 100% EtOH, 100% EtOH, 100% EtOH for 3 min each step.
4.Finish with 2 changes of xylene for 3 min each.
5.Using a Pasteur pipette, add 2-3 drops of Permount directly to the tissue on the slide.
6.Gently place a cover slip over the tissue and press out any bubbles with tweezers.
7.Allow drying completely before examining under a microscope.