I have stained the GFP mouse kidney with megalin antibody to show the proximal tubule and secondary antibody i used both mouse and rabbit TRITC to check with both of the secondary antibody show the back ground flouresence , any suggestion please.
Adult mouse kidneys have high levels of immunoglobulins in the glomerular basement membrane and also in tubules through resorption from the filtrate. Using any anti-mouse secondary is a problem. anti-Rabbits usually work although some can cross react with mouse IgGs. You can try secondary antibodies made in donkey or goat, which will work better. Alternatively, you can conjugate your fluorochromes directly to the primary antibody. This is an easy reaction available in kit form from Molecular Probes.
Thank you Gregory Dressler for your helpful comment . i feel there can be problem in blocking step as i used 1% BSA in PBS for 30 minute at room temperature. same section i did IHC stain with dako reagent and standard procedure and its working but when i did with this procedure its showing non specific binding . did http://www.rndsystems.com/ihc_detail_objectname_fluorescent_ihc_frozen_tissue.aspx
Don't forget to use 0.1% Tween 20 in all of your Ab buffers and washes. This will reduce background too.
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