Peroxidase immunohistochemistry on formalin fixed, routinely processed paraffin wax embedded renal biopsies. FIXATION
2-72 hours in 10% formal saline. The formalin fixative can be either buffered or unbuffered.
PROCESSING
Enclosed paraffin wax processor.
TRIS BUFFERED SALINE (pH 7.6)

Sodium chloride	80 g
Tris (tris hydroxymethylamine)	6.05 g
1M HCL	44 ml
Distilled water	10 litres

PROTEASE
0.1% protease [type XXIV Sigma P8038] in distilled water adjusted to pH 7.8 using 0.1M sodium hydroxide. This solution is freshly prepared and employed at 370C.

PRIMARY ANTIBODIES

Polyclonal antibody (rabbit anti-human)	Dilution Range
IgA, Ig, C3	1/4,000 - 1/6,000
IgG	1/15,000 - 1/20,000
C1q	1/5,000 - 1/6,000
Fibrinogen	1/10,000 - 1/16,000

All antibodies are supplied by Dako Ltd.,High Wycombe, UK though similar antibodies can be obtained from a number of suppliers. If an avidin biotin system is employed, an endogenous biotin blocking method is recommended and primary antibody dilutions of greater than 30,000 - 300,000 are required.

DIAMINOBENZIDINE SOLUTION
Dissolve one KEM-EN-TECH fizzing diaminobenzidine tablet, (Muratech Scientific, Aylesbury, UK) in 10ml of distilled water, filter and add one hundred microlitres of 1% hydrogen peroxide immediately before use.

IMMUNOPEROXIDASE TECHNIQUE
1. Dewax sections and take to alcohol.
2. Block endogenous peroxidase with 0.5% methanolic hydrogen peroxide for 10 minutes.
3. Take sections to running tap water.
4. Drain and transfer sections to distilled water prewarmed to 37蒀.
5. Drain off distilled water and with the minimum of delay transfer to freshly prepared protease solution (see above) at 37蒀. The digestion time is dependent on the length of fixation and the quality of the fixative. As a guide we use 40 minutes of protease digestion if the biopsy has had 6-24 hours of fixation. If the result is the plasma in the capillaries stained brown, the staining is repeated increasing the protease time by 20 minute increments. This avoids the problem of over-digestion of tissue and loss of morphology.
6. Once digestion is completed the sections are washed in running tap water for a few minutes and transferred into an incubation chamber and flooded with 2 changes of Tris Buffered Saline [TBS].
7. The slides are drained, the excess buffer removed by wiping with tissue paper and sections circled with a wax pen.
8. Sections are incubated in a 1/10 dilution of normal swine serum for 10 minutes.
9. The normal swine serum is tipped off and primary polyclonal antibody optimally diluted in TBS is applied for 40 minutes. See table for guidelines for dilution factors.
10. ash slides gently in TBS and drain.
11.Incubate in optimally diluted swine anti-rabbit Igs for 30 minutes.
12. Repeat step 10.
13. Incubate in optimally diluted rabbit perixidase anti-peroxidase (PAP) for 30 minutes.
14. Repeat step 10.
15. Incubate sections in freshly prepared diaminobenzidine solution for 10 minutes.
16. Rinse in TBS, transfer to running water and counterstain with haematoxylin.
17. Dehydrate, clear and mount.
RESULTS
Immune deposits appear brown and the nuclei blue. IgG, IgM and complement often show granular staining in the cytoplasm of the tubular epithelium.

IMAGES

1. Amyloid - AA type	2. IgA Nephropathy - IgA	3. Membranous glomerulonephritis - IgG
4. Membranous glomerulonephritis - C3	5. SLE - IgG	6. SLE - IgG

Last Modified: October 16, 1996 11:09:30 AM