Hi,
I am new to immunohistochemistry and I am trying to figure out a protocol for a ki-67 staining on a subcutaneous xenograft section.
I was hoping to use an antibody that I have on hand: http://www.bdbiosciences.com/ptProduct.jsp?prodId=653078&catyId=745877&page=product
1) It's a B56 clone, conjugated to Alexa-488. I am noticing people usually use a secondary antibody. Is there a reason for that?
2) I see there is a large variation in methods for antigen retrieval. I am planning to start with a citrate buffer, but I am not sure about the best heating method. We only have a domestic microwave that has no temperature control. is there a reason against using a water bath?
3) Sections: 5 micron; paraffin;
Hi,
A key question is to know if your Ag is resistant to your antigen retrieval procedure and still recognized thereafter by your antibody.
By the way, alexa 488 is not the most used fluorescent dye upon microscope examination, More often used in flow cytometry. But you found it on the shelf...
Good luck !
J.
The technical data sheet of your antibody doesn't say, that it can be used with formalin fixed tissue. What is the pretreatment of your specimens? Has anyone of your colleagues used it successfully on fixed specimens?
I understand, that it is a ready-to-use reagens with defined antibody-dilution. So you cannot increase the titer as you like. The only thing to amplify the signal would be prolonging the incubation time, if you don't want to use a secondary antibody with the same fluorochrome.
If your material is formalin fixed and paraffin embedded (FFPE), I would swith to another well established unconjugated antibody, that is recommended for FFPE, and take a protocol with a secondary fluorochrom conjugated antibody. The datasheet of such a primary tells you the recommended pretreatment, protocol and antibody-titer.
Hi,
normally, your antibody does work and you have not to add a secondary antibody as your primary antibody is conjugated with Alexa-488, which is a good marker in IHC.
1) the use of a secondary antibody allows you to titrate your primary antibody out to get you a better signal to noise ratio. you may have a more difficult time doing this as your antibody and fluorophore are conjugated.
2) as for heating methods, i actually use a vegetable steamer which gets to about 100 C and stays there pretty consistently. if you're using a microwave oven, you may want to play with the power levels or if you can't, just to quick blasts as to not nuke your tissue. i would actually recommend a water bath over the microwave just for temperature control. just put your tissue in a preheated coplin jar and have something to weigh it down and you should be fine.
there are multiple retrieval buffers. I use a number of them: 10 mM sodium citrate pH 6, 1M Tris Base with .5 M EDTA pH 8, and a commercial formulation called Reveal for paraffin processed tissue (mouse brain in this case). sodium citrate works pretty well for my Ki-67 that I use regularly (from thermoscientific), you may have to tweak the times. i typically retrieve for about 15 minutes using sodium citrate or 20 minutes using the tris-edta. it's also important to let you tissue cool to room temperature as to not shock the proteins (typically 10-15 minutes).
3) 5 um is fine for paraffin.
good luck!
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