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Author Topic: Indirect Immunofluorescence-3S FITC for Paraffin Secitons  (Read 2939 times)

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Offline ihcwor2

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Indirect Immunofluorescence-3S FITC for Paraffin Secitons
« on: March 12, 2003, 09:32:05 PM »
Indirect Immunofluorescence Staining Protocol for Paraffin Sections
(3 steps) (FITC)




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 before application to actual projects.

C.   Blocking Solution:
2% Normal Serum and 1% BSA in PBS:
To prepare 100 ml
Normal serum ---------- 2 ml
BSA ----------------------- 1 g
PBS ----------------------- 100 ml
Stir to dissolve.

D.   Primary Antibody:
Dilution of primary antibody is critical for success of staining, so a titration must be performed prior to application of actual projects.

E.   Secondary Antibody:
      Biotinylated secondary antibody.

F.   Streptavidin-FITC:
Purchase from Vector Labs and dilute 1:50 in PBS.

G.   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.

H.   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.

I.   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 2% normal serum in PBS.
8.   Rinse in PBS for 1x2min.

9.   Primary antibody: incubate sections with primary antibody diluted in PBS for 1 hour at room temperature (A titer must be performed prior to actual projects)
10.   Rinse in PBS 3x5 min.

11.   Secondary antibody: incubate sections with biotinylated secondary antibody in PBS for 30 minutes at room temperature.
12.   Rinse in PBS for 3x5min.

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

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

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

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


Results

1.   Positive staining -------------------------- green
2.   Nuclei ---------------------------------------- red

Indirect Immunofluorescence-3S FITC for Paraffin Secitons
« on: March 12, 2003, 09:32:05 PM »