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Author Topic: MF20 Staining of Paraffin Sections  (Read 9682 times)

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Offline jdurland

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MF20 Staining of Paraffin Sections
« on: August 13, 2005, 02:04:14 PM »
Hi,
  I'm very new to immunohistochemistry, and I've been trying to get my first protocol to work.  It involves staining paraffin sections of 4-5 dpc chicks and 8-9 dpc mice with mouse-anti-rabbit MF20, a goat-anti-mouse IgG-HRP secondary, and the DAB reaction for color.  I have been unsuccesful in getting any specific staining to work for the past month and a half, I've only produced significant amounts of background, and in some cases, there has been no staining at all.  I've tried different primary and secondary solutions as well as fluorescent secondaries instead of IgG-HRP, all to no avail.  I'm kind of at my wits end, everyone is telling me there's no reason it shouldn't work, but I can't figure it out.  If anyone could give me any advice I'd really really appreciate it.  The protocol I've been using is below:  

For embryos fixed in 4%PFA, stored in MeOH, and sectioned by standard paraffin procedure. From sections-

1)  Hemo-D X 2 (2-3 min.)



2) 100% MeOH X 2 (2-3 min.)



3) Wash slides in 1X PBS X 2 (2-3 min.)



4) Neutralize endogenous peroxidase with 0.23% H2O2 solution in water (15 min.)



5) Block with 300-500ls of 1X PBS 0.2% Triton (in the Q-Z cabinet) 2-5 % normal goat serum or NGS (in the left side of fridge): Blocking Solution. Use large Petri dishes and put down damp paper towels beneath slides, which should be elevated on wooden swab sticks so slides dont touch the water. (30 min.)
   *Note that serum in block depends on host species of 2o antibody



6) Primary antibody MF20: 300-500ls on slides
Titrate concentration in block solution or 1X PBS and NGS (goat serum, in fridge). Use Petri dishes as above or the gray slotted contraption. (3 Hrs. or Overnight)


   
7) Wash in blocking solution, 300-500ls on slides. Use Petri dishes as above or the gray slotted contraption. (30 min.)



8) 1X PBST X 1 (2-3 min.)



9) Secondary antibody Jackson sheep anti-mouse IgG-HRP 1:400: 300-500ls. On slides.
Titrate concentration in block solution or PBS and serum. (2- 3 Hrs.)


   
10) Wash with 1X PBST X 5 (2-3 min. each)



11) For HRP detection:
   incubate in standard DAB/Tris mix. (5 min.)
   incubate in DAB/Tris/H2O2 in the dark (10-20 min.).
   stop in dH2O
   Mount in Gelvatol





WORKING SOLUTIONS                  

Block:    0.2% Triton
2-5 % normal goat serum
in PBS

Volume for10 SLIDES
30% H2O2 in PBS for a 0.23% solution               1.5 ml in 200ml

Normal Goat Serum in PBS for a 5% solution            1 ml in 20 ml

Mf20 supernatant diluted 1:10 in 5% Goat Serum solution         500 l in 4.5 ml

IgG-HRP 2o diluted 1:400 in 5% Goat Serum solution         5 l in 2 ml

DAB/Tris Mix-                         10ml dH20 + 500 ul                                 DAB Stock + 500 ul
                              1M Tris pH 7.5=11ml   

Add H2O2 to DAB/Tris                     7.5 l of 30%

DAB Stock: 1 ml aliquots of Diaminobenzidine Tetrahydrochloride (10 mg/ml dH2O) (SIGMA D 5637) stored at -20? C
1X PBST=  0.1% Tween in 1X PBST

MF20 Staining of Paraffin Sections
« on: August 13, 2005, 02:04:14 PM »

Offline richard03

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MF20 Staining of Paraffin Sections
« Reply #1 on: August 13, 2005, 02:51:37 PM »
I have seen the following problems with your protocol:

1) You were using Goat Serum as blocking and reagent to dilute pirmary andn secondary antibodies. However the secondary Ab was Sheep Anti-Mouse. Note: you should used Sheep Serum as blocking if you used Sheep Secondary Ab.

2) Mouse antibody on mouse tissue can create high background and you may need special blocking reagent to solve this issue.

3) I don't see anywhere of your protocol using Antigen Retrieval method. Most of paraffin sections need this procedure.

Hope this help.

Richard

Offline jdurland

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Thanks
« Reply #2 on: August 13, 2005, 07:07:21 PM »
Richard,
  Thanks so much for your advice.  I made a mistake in saying that the secondary Ab is sheep anti mouse, it's actually goat anti-mouse.  It seems like since the DAB reaction has worked (as shown by the significant background observed in some cases), but the staining hasn't been specific, there must be something wrong with either the MF20 binding to myosin, or with IgG-HRP binding to MF20, is there anything else that might be going on?  Thanks again.  

Logan

Offline richard03

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MF20 Staining of Paraffin Sections
« Reply #3 on: August 13, 2005, 09:59:30 PM »
As I mentioned earlier that mouse ab on mouse tissue might be the cause of high background. You may also try to increase H2O2 blocking to 3% instead of 0.23%.

Richard

MF20 Staining of Paraffin Sections
« Reply #3 on: August 13, 2005, 09:59:30 PM »