I have successful experience with setotonin 2c receptors and nicotinic receptor on brain cryosection, anyway I suggest you to use hydrogen peroxide 0,3% in water for 15 min before 0,3% Triton for 15 min, as a normal immuhistochemistry...the incubation time for primary antibodies depends on the tickness of the sections, I used 3 days for 16 micrometers.

Try methanol treatment of tissue for 5 min prior to staining. Also stain with primary for at least 24 hours at 4 deg

We have used an antibody against PSD-95 (Chemicon) on mouse retina cryosections, after paraformaldehyde perfusion. Sections are delipidized with ethanol and xylene. The primary is incubated at 4 C, for 24-48 h. The procedure work fine for immunoenzymatic and immunofluorescent staining.

On this paper there is a protocol that seems to be good for such kind of staining:

http://www.jove.com/video/2270/quantifying-synapses-an-immunocytochemistry-based-assay-to-quantify?ID=2270

We also obtained some good stainings against PSD95 and vGLUT using free floating sections and immunofluorescence (72 hr of primary incubation at 4ºC). However, some antibodies do not penetrate fully into 40 microns sections (our PSD95 works highly well, lightly better using 5% Triton than 0.2% Triton) .

Good luck!

I have some experience with immuno in tissue sections, fluorescence and otherwise - but I need more information. To diagnose what's going wrong, please tell me:

is the tissue fixed?

and if so what fixative are you using?

what species and brain area?

how thick are the sections?

which antibodies, blocking buffer (*ALL* ingredients) and secondaries are you using?

And what concentrations have you tried?

Is the target epitope on the receptor intra- or extracellular.

And when you say it doesn't work - what do you see?

I assume the tissue is going to the confocal?

Thanks

David,

I agree with Anita Disney, we need some more information in order to fully help you.

With that said, I have experience with immunofluorescence directed against NR1 NMDAr subunit in 48 hour post fixed (not perfused) sections. I was able to get nice NR1 punctate labeling (1’ Ab = Rabbit anti-rat, Millipore 1:500; 2’ Ab = Alexa Fluor 488 Anti-Rabbit, Invitrogen). However, I was only able to get good signal in this tissue after antigen retrieval with pepsin (sigma).

My exact pepsin solution was:

a. 5 ml of 1N HCl

b. 20 ml of Ultra-filtered water

c. 75 mg of pepsin

You may want to try pepsin for your antigen retrieval step.

I hope this helps.

Best,

Josh

I think better you check Vincent Prevot lab paper published in J. Neuroscience

Estradiol induces physical association of neuronal nitric oxide synthase with NMDA receptor and promotes nitric oxide formation via estrogen receptor activation in primary neuronal cultures.

Coupling of neuronal nitric oxide synthase to NMDA receptors via postsynaptic density-95 depends on estrogen and contributes to the central control of adult female reproduction.

It's been a long time, but we have used the monoclonal antibody to NMDAR1 that was creates in Steve Heinemann's lab successfully on cryosections of PAF-fixed chicken retinas, so it should work like gangbusters in mammalian brain. Unfortunately it's pricey, but probably worth the expense if you have a serious need for it. See this link to one supplier, and useful references at the end including one from Heinemann's lab.

http://products.invitrogen.com/ivgn/product/320500

I have found that psd95 only gives reliable.labelling if combined with a shorter post fix 2_4h not the 16_24 that we normally use.

Sorry, I meant "PFA" (paraformaldehyde), not PAF.

Anyway, as Rohan has just written, you may have to make the fixation time very brief -- as little as 5-10 minutes in some cases that I know of. Presumably this will be the case when the epitope contains a free amino group (Lys, or N-terminus) that can be altered or blocked by the aldehyde fixative.

I tried to immunolabel PSD95 with all sorts of antigen retrieval methods, alcohol, milk powder, pepsin digestion, you name it, I tried it. In my case, I believe that pepsin+alcohol AR produces the best immunolabelling following pfa fixation. Having said that, in my experiment, I was still not convinced that my immunolabelling reflects pds95. I had doubts back then if the psd95 i got from chemicon was pure.

Anyway, pepsin digestion>alcohol fixation>then ur immunolabelling.

Thanks everybody for your answers. As pointed by Anita and Joshua, perhaps I didn't provide enough details in my previous post. I'm trying to examine composition and degree of co-localization between postsynaptic or presynaptic markers (PSD-95 or VGLUT isoforms) and ionotropic glutamate receptor subunits (NR1, NR2 or GluR subunits) in synapses by confocal microscopy. Thereby, at the postsynaptic level I am particularly concerned in achieving staining of PSD clusters. When I say it doesn´t work, I mean I don´t visualize clusters, punctate staining over surface is absent even though neurons may occasionally show dim (and perhaps non-specific) cytoplasmic staining. This proves frustrating as we never found problems to obtain good labeling for either presynaptic markers or PSD proteins of inhibitory synapses. The antibodies with which i couldn´t get satisfactory results despite all attempts are: polyclonal goat anti-PSD95 (1:200; abcam; ab12093), polyclonal rabbit anti-NR2A (1:100; Millipore. Cat. AB1555P) and polyclonal rabbit anti-NR2B (1:250; Millipore. Cat. 06-600), both the latter directed against cytoplasmic C-terminal regions. All of them work well in western blot. Antibodies against GluR subunits of AMPARs have not yet been tested. Secondary antibodies are all alexa fluor-conjugated made in donkey (dilution 1:500). In all experiments triton (0,3%) is present initially in the blocking solution and also in the cocktail of primary antibodies, but removed from the secondary antibody step on. All tests I´ve made are on neonatal rat brainstem sections (30 microns thick). Tissue is fixed by transcardial perfusion (4-5 min, 4% PFA) and later postfixed for 2-3 hours by immersion in the same fixative. The blocking buffer I'm using is PBS containing 0.3% Triton and 2,5% bovine serum albumin. In some cases and through different assays I included pretreatments intended to facilitate antigen unmasking and improve staining. Sections were pre-incubated either with formic acid (10-20 min), pepsin/HCl at 37 ºC (Fukaya and Watanabe, 2000) or with Target Retrieval Solution (DAKO) at 95-100 ºC for 30 min. None of these methods worked (in the sense denoted above).

All suggestions are welcome

Cheers

Just a commnet: In agreement with Rohan Walker , my tissue was post-fixed just for 2-3 h after intracardiac perfusion with 4% PFA (and PSD95 worked pretty well).

We have successfully used PSD95 (goat anti-PSD95 (1:200; abcam; ab12093)) following 24h postfix in 4% PFA on cryosections examining thalamic nuclei. There should be no need for antigen retrieval, just use 0.3% Triton to permeabilise tissue and wash out 16h later with 0.1% Tween.

Dear James, do you think then that 24 h postfix and wash out with Tween after primary antibody are both key steps?

I found that it worked better with a longer fixation time and generally use tween with all our immunostains, which significantly reduces background.

We have tried many different PSD95 antibodies including the goat one that was mentioned. we have found the mouse anti-PSD95 from Affinity BioReagents to be the best. It works in both paraformaldehyde fixed and methanol fixed tissues.

@ James Bourne, do you perfused and then postfix?

@Julie Lauterborn, do you know the cat# for the BioReagents antibody?

Thank you so much!

Hello,

my lab routinely performs immunstainings with PSD-95 in floating sections to look at pre-post-synapse marker colocalisation. However, we found that the PSD staining only works in acute slices, not cryo. It might be worth doing acute slices instead. Please find a link to the paper here Article Evaluation of Synapse Density in Hippocampal Rodent Brain Slices