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Author Topic: Microglia Staining Protocol (FITC) for Paraffin Sections  (Read 3435 times)

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Offline ihcwor2

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Microglia Staining Protocol (FITC) for Paraffin Sections
« on: March 12, 2003, 09:42:18 PM »
Microglia Staining Protocol (FITC) 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.   Antigen Retrieval Solution:
10mM Sodium Citrate Buffer (The most commonly used antigen retrieval reagent):
To prepare 1000 ml,
Sodium citrate ------------- 2.94 g
Distilled water ------------- 1000 ml
Adjust pH to 6.0

Selection of antigen retrieval techniques is crucial for successful staining of paraffin sections. Different antibodies may require different antigen retrieval techniques and need to be tested prior to application of actual projects.

C.   Blocking Solution:
1% BSA in PBS:
To prepare 100 ml
BSA ----------------------- 1 g
PBS ----------------------- 100 ml
Stir to dissolve.

D.   Biotinylated lectin RCA-1:
      Concentration range: 5-25 ug/ml.

E.   Streptavidin-FITC Reagent

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.

       
Procedure

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: (Select an appropriate antigen retrieval technique and it depends on antibodies used).
6.   Rinse sections in PBS for 1x5min.

7.   Blocking: incubate sections with 1% BSA in PBS to block non-specific binding.
8.   Rinse in PBS for 1x2min.                                                                        

9.   Lectin RCA-1: Incubate sections in biotinylated lectin RCA-1 for 1 hour at room temperature.
10.   Rinse in PBS for 3x5min.

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

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

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

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


Results

1.   Microglia ---------------------------------- green
2.   Nuclei -------------------------------------- red

Although endothelia cells and blood cells reacted with RCA-1, they were easily distinguished morphologically from microglia. Astrocytes, oligodendrocytes, and neurons did not react with RCA-1.

Microglia Staining Protocol (FITC) for Paraffin Sections
« on: March 12, 2003, 09:42:18 PM »