background staining in immunofluorescence - (Apr/21/2005 )
Hi, when i stain human brain sections (10 micron) for double labelling i get a lot of background staining.. and also i feel that both the primary antibodies pick up at similar sites... can anyone help me with this . especially the background staining problem
what kind of controls do you have?
Do your primary antibodies have the same isotype?
Do you have an isotype control? Do you have a control staining with the second antibody alone?
Do you block your sections with for example BSA or something?
If your primary antibodies occur at similar sites: should this be impossible? -If so: your staining is maybe not specific.
Great questions bomber -- extremely pertinent ... one more question:
What fix if any do you use? Makes a HUGE difference.
Did you try NaBH4 treatment, it works well to remove auto-fluorescence.
No. the pry Ab do not have the same isotype.. i have tried fixing the tissues in 10% buffered formalin .. and also a seperate one in 4 % buffered formalin.. i do not use BSA.. and the big.. major problem.. i never get the neuronal picture even with CHAT staining
Are these paraffin or frozen? Either way you can expect huge autofluorescence from formaldehyde based fixations. You can block this using a variety of methods including the one mentioned above (sodium borohydride), as well as glycine treatment, ammonia alcohol and sudan black.
10 um by the way is very thick for paraffins and getting thick for frozen, can you cut thinner?
Also, I would not recommend you use 4% neutral buffered formalin at all - stick with the prepared 10% NBF. Diluting it might only cause problems in fixation.
Can you provide a more detailed protocol of what you did and I will try to help?
and if it is possible to use with monocyte derived DC??
thank you in advance