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Author Topic: PCNA Immunofluorescence Protocol for Paraffin Sections  (Read 2819 times)

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Offline ihcwor2

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PCNA Immunofluorescence Protocol for Paraffin Sections
« on: March 15, 2003, 02:24:10 AM »
PCNA 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.   Antigen Retrieval Solution:
10mM Sodium Citrate Buffer:
To prepare 1000 ml,
Sodium citrate ---------- 2.94 g
             Distilled water ---------- 1000 ml
             Adjust pH to 6.0

C.   Blocking Solution:
2% Normal Horse Serum in PBS:
To prepare 100 ml
Normal horse serum ------- 2 ml
PBS ---------------------------- 100 ml
Mix to dissolve.

D.   Primary Antibody:
Mouse anti-PCNA (Santa Cruz Biotechnology, Cat# sc-56). Optimal dilution 1:2000 in PBS.

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

F.   FITC-Streptavidin Reagent:
Vector Laboratories, Cat# SA-5001. Optimal dilution 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: Use steamer-citrate buffer antigen retrieval method.
6.   Rinse sections in PBS for 1x5min.

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

9.   Primary antibody: incubate sections with mouse anti-PCNA diluted 1:2000 in PBS for 1 hour at room temperature.
10.   Rinse in PBS 3x5 min.

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

13.   Incubate sections in FITC-streptavidin 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.   PCNA positive staining ---------------------- green
2.   Nuclei --------------------------------------------- red

PCNA Immunofluorescence Protocol for Paraffin Sections
« on: March 15, 2003, 02:24:10 AM »