Immunofluorescence on mouse tissues - (Jul/09/2008 )

Hi guys,

I just started my new job and am coming across all these different techniques. And of course I'm going to my resource for the past several years for several things!
I'm trying to set up a new project for confocal microscopy and have never used it before. Sure I have done some immunology, WB and FACS staining, but never this...
My biggest question is what do you test out when you're starting a new antibody?
So basically controls?...
Just to let you know that I did think about this before I started the topic and that I did do some research...
Controls, of course positive control specific for the ab of choice, also negative control.
I'm going to use indirect staining, so I need a control of the whole staining protocol w/o the primary and 1 w/o the secondary ab. Could I just replace the primary and secondary w/ PBS?
Since I'm going to eventually use it on tissues I'm planning on doing a pilot with the different tissues I'm planning to use the ab on.

Also... Because I'm limited in different channels for the confocal microscope I would need different slide of the same tissues for different combinations of abs. Any good way of doing this or any specific way of what combinations to choose and what not? Also in general do you have 3 different tissue slides to get your triplicates? Because that would mean I need a whole lot of slides from each tissue and I'm not sure how many I can get? (I know this is where my inexperience comes in!)

So this is what I'm thinking, just let me know what I'm missing (because I'm sure I'm missing something!), still need to address and if you have general good tips/ideas, everything is welcome....

Thanks so much!

Confocal Microscopy was basically my life in grad school...

I can't say for sure but I think you might be making it more difficult on yourself than it needs to be...

Let's try to simplify this...

I assume one set of tissue will be exposed to some experimental condition. So, you'll have
-Control Tissue
-Experimental Tissue

Labeling Control Tissue

+ control - relevant primary antibody / relevant secondary antibody
- control - relevant secondary antibody alone
--------or - irrelevant primary antidody / relevant secondary antibody

I never use the relevant primary antibody for the negative control, it's a waste of money as relevant primary antibodies are expensive.

Labeling Experimental Tissue
Experimental - relevant primary antibody / relevant secondary antibody


So you have three groups:

Positive Control - which shows what the untreated cells looks like
Negative Control - which shows background fluorescence or non-relevant fluorescence

Experimental Control - which is labeled exactly as the positive control is so you can see what changes are brought about...

You'll probably soon find out that with confocal microscopy people seldom do a negative control... Confocal microscopy isn't really for Quantification so you don't worry about mathematically subtracting the background, you simply do it with the microscope controls...

You know, after reading this, I don't think I've told you anything you don't already know. If that's the case I apologize... However, I hope this helps...

Again, I do know a good bit about confocal microscopy, I'd be happy to answer any questions you might have, just send me an email...

Good luck

Thanks, I probably will!