PSD95 staining - (Dec/01/2006 )
Hi,
I'm also trying to stain for PSD95 on my primary neuronal culture. For some reason, I can not detect signal at all. I'm also expecting puncta staining, but it does not seem to work well. My neurons are plated at a density that synapse and denrities have form, etc. Can anyone help me with the protocol please? I fix in 3.7% formaldehyde for 10 min, then block with BSA, dilute primary in BSA overnight and secondary stain for 1hr in BSA.
Please help. Thank you.
--Jessica
I'm also trying to stain for PSD95 on my primary neuronal culture. For some reason, I can not detect signal at all. I'm also expecting puncta staining, but it does not seem to work well. My neurons are plated at a density that synapse and denrities have form, etc. Can anyone help me with the protocol please? I fix in 3.7% formaldehyde for 10 min, then block with BSA, dilute primary in BSA overnight and secondary stain for 1hr in BSA.
Please help. Thank you.
--Jessica
is it para-formaldehy or really formaldehyd?; try to reduce to 2% in the case of para-formaldehyd or alternative fixation methods such as methanol or aceton; what is the secondary stain?
It's 3.7% paraformaldehyde. I will try the 2% fixation. Do you know what's the difference between methanol fixation and this one? Because for most papers I researched, paraformaldehyde seems to work fine. My secondary is Alexa 488. I think the problem maybe primary PSD955 antibody dilution factors. Can anyone tell me what dilution they use and in what percet of BSA please? Thanks!!
--Jessica
--Jessica
I we also work with secondary Ab Alexa 488 but not with neurons; nevertheless, when I optimize IHC with new Ab or new cells, I try fixation with para-formaldehyd (4 or 2%), or methanol or aceton;
beside fixation, permeabilization (some do before, some after fixation) is necessary; contains mild detergents such as saponin; I once tried to stain w/o permeabilization but I failed; so if you do not permeabilize, it is very recommended!
I'm also trying to stain for PSD95 on my primary neuronal culture. For some reason, I can not detect signal at all. I'm also expecting puncta staining, but it does not seem to work well. My neurons are plated at a density that synapse and denrities have form, etc. Can anyone help me with the protocol please? I fix in 3.7% formaldehyde for 10 min, then block with BSA, dilute primary in BSA overnight and secondary stain for 1hr in BSA.
Please help. Thank you.
--Jessica
Dear Jessica,
A lot of proteins of the post-synaptic density are difficult to visualize by immunofluorescence. I know that for many of these proteins ice-cold methanol fixation works better than PFA. I do not know the exact reason for this, but I guess that you may need some kind of unmasking the very dense structures. Unfortunatelly we do not have a PSD 95 antibody that works well for immunofluorescence I could offer, but as far as I know sternberger monoclonals has one.
Good luck,
Henrik
Dr. Henrik Martens
Synaptic Systems GmbH
Rudolf-Wissell Straße 28
D-37079 Göttingen
Tel. +49 551-50556-358
support@sysy.com
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Synaptic Systems Gesellschaft für neurobiologische Forschung, Entwicklung und Produktion mbH
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As far as I remeber I used to use the mixture of methanol and aceton (ice cold) and for short time as 10 min. You can also try to start with formaldehyd for 10 min and then continue with methanol/aceton. Good luck!