Hi Thorsten,

We regularly perform immunofluorescence staining on mouse brain tissue and have found that although there is some background fluorescence mainly in the FITC channels this can be resolved by using the correct blocking buffers and trying to minimize the use of mouse antibodies (use of mouse antibodies on mouse tissue can necessitate additional blocking steps). I've attached one of our basic immunofluorescence protocols for use on cryosectioned brain tissue. 

Good luck.

Hi Thorsten,

Is it a specific antibody that makes it problematic for you?

You can find here a basic protocol that worked for all the antibodies we used in this study, so that shows you this protocol works with multiple antibodies easily:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3010824/

Can you give more technical details and perhaps load images?

hi

be careful Fat in the brain tissue leads to Autofluorescence or background ( dye seems as yellow not really green ) .

1- you can used irrelevant antibody in your test as negative control to normalized photos .

2- check Acetone Fixation method for cryosectioned brain tissue . ( 2 min slides in -20c acetone and then dry for 30 min in 4c , so complete drying in RT for 15 min )

good luck

Hi Thorsten!

Protocol for immunofluorescencent labelling of free floating brain slices (transcardially perfused rats, 4% formaldehyde), stored in cryoprotectant at -20°C (Tratnjek et al., 2016. Eur J Neurosci. 2016. Apr;43(7):885-98, https://www.ncbi.nlm.nih.gov/pubmed/26750488):

Rinse the sections in KPBS/PBS tree times for 5 minutes each. For antigen retrieval (optional) incubate slices in Na-citrate buffer (30 min, pH=8.5-9 at 80°C) or in 0.5% NaBH4 in KPBS (prepare just before the use, incubate 15 min, room temperature). Rinse with KPBS 3 times 5 minute each. Block sections with blocking solution containing 4% normal serum, 1% bovine serum albumin (BSA) and 0.1% Triton X-100 in KPBS for 1 h at room temperature. Next, incubate brain slices with the primary antibodies diluted in blocking solution for 1-2h at room temperature and then overnight at 4 °C. Wash sections with KPBS containing 0.02% Triton X-100 6 times for 10 minutes each. Incubate slices with the secondary antibodies diluted in KPBS containing 1% BSA and 0.02% Triton X-100 for 1 h at room temperature. Wash with KPBS 4 times 10 minutes each. After the incubation, immerse slices into 0.1% Sudan Black B (Sigma) in 70% vol/vol ethanol for 5 min to suppress lipofuscein background autofluorescence. Rinse with KPBS 3 times 5 minute each. Finally, mount the sections and coverslip using antifade reagent such as Prolong Gold.

Hope this helps.

Hi Thorsten, did you use paraffin sections or frozen sectons? Depending on your primary antibodies (mouse), anti-mouse- ab could be responsible for the high background staining, Negativ controls can help to find out wheather this is the reasen therefore.   If you need to work with mouse on mouse  abs use in the blocking solution high concentrations of normal serum  as well as in your delution medium of the primary ab (20-30%). Autofluorescnence could be an explications of unspecific staining  in your sections. In that case I recommand  to check  non stained sections under the microscope. Sudan Black stain reduces autofluorescence in frozen sections Finally if it is hard to distinguish real immuno signal from background staining and you need no double labeling I recommand  DAB stain of peroxidase labeled secondary antibodies or ABC/Streptavidin. Good luck!