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Microglia Staining Protocol
Description:
This method is for demonstration of microglia
in brain tissue. Although endothelial cells and blood cells reacted
with RCA-1, they were easily distinguished morphologically from
microglia. In addition, astrocytes, oligodendrocytes, and neurons
did not react with RCA-1, so this method stains microglia
specifically.
Fixation: Formalin-fixed, paraffin embedded sections, and acetone fixed, frozen sections.
Positive Controls: Brain tissue.
Solutions and Reagents:
Lectin RCA-1, Biotinylated:
Vector Laboratories, Cat# B-1085. Optimal dilution 1:1000.
Detection Reagent:
HRP-Streptavidin (Vector Laboratories, Cat# SA-5004). Optimal
dilution 1:500
Procedure:
1. Sections to distilled water
2. Epitope Retrieval: Not needed.
3.
Rinse
sections in 2 changes of PBS-Tween 20, 2 minutes each.
4.
Peroxidase Blocking:
incubate sections in 3% H2O2 in PBS for 10 minutes to block
endogenous peroxidase activity.
5. Rinse in PBS-Tween 20 for 3x2 min.
6.
Serum Blocking:
incubate sections with 1% BSA in PBS for
30
minutes to block non-specific binding of immunoglobulin.
7. Rinse in PBS-Tween 20 for 3x2 min.
8. Lectin RCA-1: incubate sections in Biotinylated Lectin RCA-1 (Vector Labs) 1:1000 diluted in PBS for 1 hour at room temperature.
9.
Rinse in
PBS-Tween 20
for 3x2 min.
10.
Detection:
incubate sections with HRP-Streptavidin diluted in PBS for 30
minutes at room temperature.
11.
Rinse in
PBS-Tween 20
for 3x2min.
12.
Chromogen/Substrate:
incubate sections in DAB solution for 5-10 minutes.
13.
Rinse in
distilled water briefly.
14.
Counterstain
with Gill's or Mayer's hematoxylin if desired.
15.
Rinse in
running tap water for 5 minutes.
16.
Dehydrate
through 95% ethanol for 2 minutes, 100% ethanol for 2x3min.
17. Clear in xylene for 2x3min.
18. Coverslip with permanent mounting medium.
Results:
Staining pattern: Microglia, blood vessels (endothelial cells)
Notes:
1. One may need to block endogenous biotin activity for certain tissues such as kidney, liver, prostate, colon and gut, which may contain endogenous biotin.
2. For frozen sections, snap frozen fresh tissues in isopentane pre-cooled in liquid nitrogen, embedded in OCT compound in cryomolds. Cut 4-8 um cryostat sections and mount on superfrost plus slides. Store slides at - 80°C until needed. Before staining, air dry slides at room temperature for 30 minutes and fix in ice-cold acetone for 5 minutes. Air dry for another 30 minutes. Then start from step 3 for routine immunostaining.
References:
Mannoji H, et al (1986) A specific
histochemical marker (lectin Ricinus communis agglutinin-1)
for normal human microglia, and application to routine
histopathology. Acta Neuropathol (Berl). 71: 341-343.