Methods and Techniques Discussion => Immunohistochemistry (IHC) => Topic started by: ihcwor2 on March 15, 2003, 01:36:24 AM

Title: Beta-Amyloid Immunofluorescence Method for Paraffin Sections
Post by: ihcwor2 on March 15, 2003, 01:36:24 AM
Beta-Amyloid Immunofluorescence Staining Protocol for Paraffin Sections

Solutions and Reagents

A.   10X Phosphate Buffered Saline (PBS):
To prepare 1 liter,
Na2HPO4 ------------------ 10.9 g
NaH2PO4 ------------------ 3.2 g
NaCl ------------------------ 90 g
Distilled water ----------- 1000 ml
Mix to dissolve and adjust pH to 7.4

B.   Blocking Solution:
2% Normal horse serum in PBS:
To prepare 100 ml
Normal horse serum -------- 2 ml
PBS ----------------------------- 98 ml
Mix to dissolve.

C.   Primary Antibody:
Mouse anti-beta-Amyloid (Novocastra, Cat# NCL-B-Amyloid). Optimal dilution 1:100 in PBS.

D.   Secondary Antibody:
      Horse anti-mouse IgG (H+L), biotinylated (Vector Laboratories, Cat# BA-2000). Optimal dilution 1:400.

E.   FITC-Streptavidin Reagent:
Vector Laboratories, Cat# SA-5001. Optimal dilution 1:50 in PBS.

F.   PI Stock Solution (1mg/ml or 1.5 mM):
To prepare, add 1 mg PI (Propidium Iodide) to 1 ml distilled water. Store stock solution at 4 C (or aliquot and store at 20 C), protected from light.  When handled properly, solutions are stable for at least six month.

G.   RNase A Stock Solution (1mg/ml):
To prepare, add 1 mg RNase A to 1 ml distilled water. Aliquot and store at 20 C freezer.

H.   PI Working Solution (1 ug/ml PI and 10 ug/ml RNase A in PBS):
To prepare, add 2 ul PI Stock Solution and 20 ul RNase A Stock Solution to 2 ml PBS.


1.   Deparaffinize sections in xylene for 3x5min.
2.   Hydrate with 100% ethanol for 2x5min.
3.   Hydrate with 95% ethanol for 2x5min.
4.   Rinse in distilled water.

5.   Antigen Retrieval: Treat tissue sections with 98-100% formic acid for 3 minutes. Wash the sections in distilled water briefly and rinse in PBS for 2x5min.

6.   Blocking: incubate sections with 2% normal horse serum in PBS for 20 minutes
7.   Rinse in PBS for 1x2min.

8.   Primary antibody: incubate sections with mouse anti-beta-Amyloid antibody diluted 1:100 in PBS for 1 hour at room temperature.
9.   Rinse in PBS 3x5 min.

10.   Secondary antibody: incubate sections with biotinylated horse anti-mouse IgG diluted 1:400 in PBS for 30 minutes at room temperature.
11.   Rinse in PBS for 3x5min.

12.   Incubate sections in FITC-streptavidin for 30 minutes, protecting the slide from light.
13.   Rinse 3x5min in PBS.

14.   Counterstain with PI Working Solution for 20 minutes at 37 C.
15.   Rinse 3x5min in PBS.

16.   Coverslip with aqueous mounting medium and seal with nail polish.

17.   Observation using a fluorescence microscope with appropriate filters. Store slides in the dark at 4 C.


1.   Senile plaques ---------------------------- green
2.   Nuclei ---------------------------------------- red