Immunoflourescence of Frozen Brain Tissue - (Sep/26/2014 )

I'm trying to stain a membrane protein in frozen sections of brain tissue. Histology is new to me, but there are members of our lab whom are wizards when it comes to muscle tissue. I've run into issue with high background, and I can't figure out why. Here is the protocol:

Tissue Extraction and Processing

1) Brain tissue was removed, and fixed in 4% paraformaldehyde-PBS. Snap frozen in OCT, stored at -80C.

2) Sectioned tissue at 10um. Placed on slides, stored overnight at -20C

Staining Procedure

1) Removed slides from freezer. Allowed to warm at room temperature for 15 minutes.

2) Washed in PBS 3 times, 5 minutes each

3) Permeabilized with 1% Triton-X-PBS, 20minutes at room temperature

4) Washed in PBS 3 times, 5 minutes each

5) Incubated with 10% Normal Goat Serum, 15 minutes at room temperature

6) Primary antibody, rabbit polyclonal; 1:50, 1:25 (tried both dilutions separately); 60 min @ 37 C

7) Washed in PBS 3 times, 5 minutes each

8) Incubated with 10% Normal Goat Serum, 15 minutes at room temperature

9) Secondary antibody. Goat anti-rabbit IgG-FITC. Tried 1:300, 1:500, and 1:1000. 30 min at 37C

10) Washed in PBS 3 times, 5 minutes each

11) Added DAPI, cover slide, and viewed.

My negative controls (processed the same as above, except PBS-BSA was added in place of Primary antibody), look the same as my samples. There is a lot of background, with the occasional brighter green. The brighter green does seem to be localized to cells, which is extra confusing.

When there isn't a lot of signal, will the microscope increase its "signal" to try to find the "positive" areas? So if there wasn't any FITC on my samples or negative control, then you would expect to see a lot of green background? I tried adjusting the exposure, but I can't seem to get rid of the green background on my samples or negative controls.

Anyone have any idea what's going on? Is this a microscope issue, and both my slides are just negative for the protein? My lab mate gets nice muscle images, free of background, but he's having trouble communicating (language barrier).

-djvan-

Hi djvan,

You sounded like me when I first started doing immunofluorescence staining of brain cryosections. I have learned a lot since then and hope that some of my suggestions will help you solve your problems.

Firstly, switch to TBS instead of PBS.

You didn't mention if you did transcardial perfusion of your mice (I'm assuming you are working with mice) prior to brain isolation. If you didn't do perfusion, the mouse serum/blood cells in the brain are the most likely cause of your background fluorescence. Red blood cells autofluoresce in the green channel. Also, if you didn't clear the mouse serum, the immunoglobulins in it will pick up the secondary anti-mouse antibody if you are using it. So, make sure you do proper transcardial perfusion before brain isolation.

In my experience, goat secondary antibodies are always very "dirty" and I avoid them like the plaque. Donkey ones on the other hand are much better. I use only donkey secondary antibodies for all my stainings.

If you think you see weak positive staining but the background is interfering, you can increase the contrast by increasing "offset" value during image acquisition.

Finally, there is a notorious cause of autofluorescence that is commonly encountered in old brains. It is caused by lipofuscin, a breakdown product that accumulates in neurons. Lipofuscin autofluorescence appears as blotches and shows up in all channels including DAPI. To get rid of it, you need to treat stained samples with CuSO4 dissolved in NH4Cl.

Additional tip:

FITC photobleaches easily and you should take care not to expose your samples to light during and after staining. It also tends to bleach quite fast under epifluorescence or laser. If you have the option to buy a replacement, get Alexa Fluor 488.

Hope this helps. Good luck.

~neuropath~

-neuropath-