Author Topic: Indirect Immuno-AP Method (BCIP/NBT) for Paraffin Sections  (Read 5007 times)

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Offline ihcwor2

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Indirect Immuno-AP Method (BCIP/NBT) for Paraffin Sections
« on: March 12, 2003, 08:14:08 PM »
Indirect Immuno-AP Method (BCIP/NBT) for Paraffin Sections

Solutions and Reagents

A. TBS Buffer (10mM Tris-HCl, 150mM NaCl, pH 7.5):
Tris-HCl (MW 157.60) --------------------- 1.58 g
NaCl (MW 58.44) ---------------------------- 8.77 g
Distilled water -------------------------------- 1000 ml
Mix to dissolve and adjust pH to 7.5

B. AP Buffer (100mM Tris-Cl, 100mM NaCl, 5mM MgCl2, pH 9.5)
Tris-HCl (MW 157.60) --------------------- 15.76 g
NaCl (MW 58.44) ---------------------------- 5.84 g
MgCl2 (MW 203.31 -------------------------- 1.02 g
Distilled water --------------------------------- 900 ml
Mix to dissolve. Adjust pH to 9.5 and then bring total volume to 1 liter.

C. NBT Stock Solution: 5% NBT (nitro blue tetrazolium) in 70% dimethylformamide and store in aliquots at 20 C.

D. BCIP Stock Solution: 5% BCIP (5-bromo-4-chloro-3-indolyl phosphate) in 100% dimethylformamide and store in aliquots at 20 C.

E. AP Staining Solution (0.33mg/ml of NBT, 0.165mg/ml BCIP in AP Buffer):
NBT stock solution ----------------------- 66 ul
BCIP stock solution ---------------------- 33 ul
AP buffer ----------------------------------- 10 ml
Mix well and use immediately. (Alternatively, BCIP/NBT tablets can be obtained from Sigma (Cat# B5655).

F.   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.

G.   Levamisole Solution

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

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

J.   Secondary Antibody:
Alkaline phosphatase conjugated secondary antibody

K.   BCIP/NBT Reagent

       
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 TBS for 1x5min.

7.   Blocking: incubate sections with 2% normal serum in TBS to block non-specific binding of secondary immunoglobulin.
8.   Rinse in TBS for 1x2min.                                                                        

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

11.   Secondary antibody: incubate sections with alkaline phosphatase conjugated secondary antibody in TBS for 30 minutes at room temperature.
12.   Rinse in TBS for 3x5min.

13.   BCIP/NBT: incubate sections with AP staining solution containing Levamisole for 20-30 minutes to several hours.
14.   Rinse in distilled water 2x5min.

15.   Counterstain with nuclear fast red if desire.
16.   Rinse in distilled water 2x5min.
17.   Dehydrate through 95% ethanol for 5min, 100% ethanol for 2x5min.
18.   Clear in xylene for 2x5min.

19.   Coverslip with mounting medium.


Results

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

Indirect Immuno-AP Method (BCIP/NBT) for Paraffin Sections
« on: March 12, 2003, 08:14:08 PM »