[Histonet] probelm with an immunofluorescence of mammary gland on paraffin sections

[janick.jean-marie]

Hi fellows !!
I have a problem with an  immunofluorescence  of mammary gland on paraffin sections 
Indeed , I have not a specific immunoflurescence marking ...

My protocol :

Deparaffinization stage :

? Xylène , three times for  min each 
? 100% EtOH  twice for 5 min 
? 95% EtOH  once for 5 min 
? diH2O or PBS 1X 

Antigen Retrieval stage :

                                          Citrate buffer 
                                              (10mM)
Reagents:
a.    Citric Acid, monohydrate, granular
    Storage: Room Temperature
b.    Filtered distilled water
c.    10M Sodium Hydroxide
    
Preparation:    10 mM Citrate Buffer
  a. Dissolve 0.525g citric acid monohydrate in 250mL of distilled H20 (dH2O)
  b. pH to 6.0 +/- 0.3 with 10M NaOH 
       Storage:  Room Temperature

-Preheated container water at 94°C and place the container of Citrate buffer in the bath.
-Put the slides in the citrate buffer for 45 min 
-Remove the Citrate Buffer container with the slides , out of the bath and let’s cooling at RT 
 for 20 min
-Plunge the slide in PBS (or H2O) for stop the reaction 


? Begin the Immunofluorescence Protocol

1. Antiboby A? against Beta-GAl(1/500) 
2. 1 hour in humid chamber 
3. Bath of PBS 1x  for 15 min
4. flurorescent secondary antibody (1/300)  (Rhodamin)
5. humid chamber for 30 min 
6. wash with PBS 
7.  add mowiol for mountig scale
8. Stock at  -80°C

.... I don't knows what's wrong ....

maybe i can use a wash of PBS-tween and before the step with Antibody A? , i can make a bath of milk(2%) for 1 hour ...

Wath do you mean about my protocol and suggestions ?

Another idea ?


 THANKS !!!

Janick JEAN-MARIE
CRLC INSERM 

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