rev: October 6, 1998

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Sample Histochemistry fixation & Saponin Protocol  

(for intracellular cytokine staining and paraffin embedded sections after high temp release-for saponin permeabilization)

Saponin Method:

Dulbecco's PBS (without Mg2+ or CA2+)

1% heat inactivated FCS (or BSA-do not use animal serum with Biotin:Streptavidin detection systems due to endogenous biotin levels often found in serum or some skim milk preparations)

0.1% w/v sodium azide

0.1% w/v saponin (Sigma cat#S-7900)

Adjust pH buffer to 7.4-7.6 and filter

see ref:

1)Sander, B., J. Andersson and U. Andersson, 1991:Assessment of cytokines by immunofluorsecence and the paraformaldehyde-saponin procedure:Immunol Rev 119:65-93

2) Anderson, U. and J. Andersson 1994. Immunolabelling of cytokine producing cells in tissues and suspension. In Cytokine Producing Cells eds. D. Fradelizie and D. Emelie. INSERM, Paris. 32-49.

for intracellular cytokines, stop golgi process with with either monesin (Sigma cat#M-5273) or Brefeldin A (Sigma cat#B-7551)
-ref#Nature 373:255-257

-Prussin C and Metcalfe DD. Detection of Intracytoplasmic Cytokine Using Flow Cytometry and Directly Conjugated anti-Cytokine Antibodies. Journal of Immunological Methods, 188, 117-128 (1995).

-J. Immunol Meth 159:197-207

Sample Initial fixation:

the following are sample fixation methods for histochemistry:

if on tissue culture cells:

a) 10 minutes in 1% formalin in PBS (keep wet) -OR-

b) 5 minutes in -10 DEG C methanol, air dry -OR-

c) 2 minutes in cold acetone, air dry

then rinse in three changes of PBS, incubate for 5-10 minutes in 0.1-1% hydrogen peroxide in PBS to quench any endogenous peroxidase activity (optional)

-or if on frozen tissue sections

a) 4-8 micron thick sections, frozen in isopentane precooled in liquid nitrogen, embedded in OCT compounds in cryomoids and stored at -70DEG C.

b) allow frozen sections to come to room, temp (30 minutes 

c) fix slides in cold acetone for 10 minutes, keep refrigerated (or use other fixation methods). Rinse in three changes of PBS

d) incubate for 5-10 minutes in 0.1-1% hydrogen peroxide in PBS to quench any endogenous peroxidase activity (optional)

certain tissue may then also require treatment with pepsin or with saponin (to permeabilize the membrane to allow the antibdy to penetrate into the intracellular region

(incubate for 30 minutes in 2-10ug/ml pepsin in 0.01N HCL buffer, pH 2.5 at room temp, wash slides several times in distilled water

-or incubate for 30 minutes in 2-10ug/ml saponin in distilled water at room temp, wash at least three times with PBS

-block for 1.5 hour in 1.5% normal blocking serum in PBS, Wash/aspirate, keep slides from drying out for all latter steps.

-continue with staining titer 1, 0.5, 0.1ug/slide diluted in blocking serum in PBS overnight at 4 DEG C or 2 hours at room temp.

-Wash 3X with PBS for 5 minutes each

-visualize with detection method of choice (recommend Vector Elite  (tm) or equivalent Streptavidin:Biotin methodology).

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