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Author Topic: IHC on sputum cytospins  (Read 6262 times)

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Offline jonnieplumb

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IHC on sputum cytospins
« on: December 22, 2005, 10:24:00 AM »
Hi everyone and Merry Christmas
I am working on sputum cytospins but my negative slide (ie primary ab omitted) is showing signs of staining following both Ipx and AP detection.
Any ideas?
I've been fixing in acetone, would using methanol or acetone-methanol (1:1) help?
I've blocked endogenous biotin, peroxidase and phophatase

IHC on sputum cytospins
« on: December 22, 2005, 10:24:00 AM »

ikirbis@onko-i.si

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immuno on sputum
« Reply #1 on: December 28, 2005, 02:47:09 AM »
I guess that fixation in other fixative wouldn't help, but you can probabaly get rid of non specific staining with some additional steps in cytospin preparatory technique, suspending the sample in good cell medium (PBS with some bovine serum albumine) or some other commercially available cell medium will probably give you better results
merry christmas

Offline richard03

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IHC on sputum cytospins
« Reply #2 on: December 28, 2005, 10:03:08 PM »
You may want to consider the secondary antibody binding to the cell unspecifically. Did you do normal serum blocking? What did you use?

Richard

Offline jonnieplumb

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IHC on sputum cytospins
« Reply #3 on: January 05, 2006, 06:36:02 AM »
Happy New Year
Thank svery much for your comments.
I have repeated with acetone/methanol fixation with extra PBS washes (Pre and post fixation) and seems to have worked well.
I had been using mouse anti-human Abs detecting using BRAM secondary so was blocking in 5% NRS.
Cheers
J

IHC on sputum cytospins
« Reply #3 on: January 05, 2006, 06:36:02 AM »