High background in mouse retina wholemount staining

madsmad · **madsmad**

Hello there,
I'm doing wholemount double staining of mouse retina with Brn3a and UF006 (both goat and rabbit polyclonal respectively) antibodies. The problem is, I get very high background which do not permit me to see the cell signals. Could anyone please guide me with a good protocol?
Thanks

Carl · **Carl**

You do not give details of your method.
Are you getting a good signal with your 2 Abs BUT there is too much background?
If yes, what microscope sytem are you using for examination?
For wholemount only a confocal ( or Apotome) system will be suitable.

Do you know if your two proteins are expressed in the retina?
Do you know if your two Abs will work on wholemounts?
Adult or embryonic retina?
What other Abs have you used, to indicate that your method works?
We only have your word that your technical expertise is good.
Thus, some images of your results would be very helpful

Adult retina is difficult for IF labelling as wholemount, because the tissue is very dense.
Using 1% detergent or even DMSO can make the tissue more permeable.
Long detergent-containing washes between all steps, are required.
Short fixation time, if using Formaldehyde -based fixation, is essential.

All Imho....

madsmad · **madsmad**

Thank you Carl. Here's the protocol that I follow.

1. Eyes are fixed in 4% paraformyldehyde for 4 hours and then washed in PBS 3x to use it later.
2. After the retinas are dissected out, they are permeabilized in 0.3% Triton X-100 for 1 hour
3. Blocking of non-specific staining with a blocking buffer containing BSA 3%, NaN3 0.1%, Tween20 0.05% and Donkey serum 5% for 24 hours.
4. Incubation with primary antibodies in the blocking buffer for 72 hours
5. Washing with PBS containing 0.05% Tween-20 3x for 20 minutes and then for 24 hours.
6. Incubation with secondary antibodies for 24 hours.
7. Washing with PBS containing 0.05% Tween-20 3x for 20 minutes and then for 24 hours.
8. Mounting the retinae on slide with PBS:Glycerol (1:1).
Our microscope is Nikon Optiphot 2. We don't have confocal microscope.
Both antibodies do work but the problem is high background. I don't want to use different software techniques to decrease the background.
Here's an image of ganglion cells. I have reduced the URL size for easy access. Its mouse retina at P17.

Image here

http://bit.ly/lQ13dm

Thanks

Carl · **Carl**

The image you linked has no background noise, imho....it is just low signal, thus there is a slight red background?
Do you use Photoshop, for eg, to manipulate globally?
Imho, you will benefit .
Sure, anyone please help/contradict me on this.

Protocol looks good but, you change TWEEN concentration between 0.1% and 0.05%...why?
Also, you do not seem to add TWEEN to Primary/secondary Ab?
Drop the dead Donkey serum Wink

Unless you can justify using it?

Add Hoechst or DAPI to your secondary Ab so we can see nuclei.
Mount your retinae on slides using an anti-fade mountant, not PBS-Glycerol ( sure, many do not do this and argue that there is very little fading...however, what fluorochromes are you using??)

Re microscopes: sorry...if you are doing whole-mount IF there is no way to get good/representative images of the whole depth of the tissue if you are using conventional IF microscopy, imho.
Why?
Because you only capture a single focal plane but...also capture all out of focus focal planes so...image is blurred.
Confocal will cut out of focus light.
Sure, you then need to do Z axis image capture.
Send me your image, please, as I cannot save and open it on my PC.
Thanks.
Carl

madsmad · **madsmad**

In my opinion, its the best signal I've got so far. How can I increase the signal and decrease the red background at the same time?
Once I tried fixing the retina in cold acetone at -20 °C for 10 minutes. Got comparatively better signals and low background but the problem is, I can't dissect the retina afresh because retina liquifies during dissection. I have no other choice than to dissect it post PFA fixation. I use photoshop as well but I don't get good images. The problem is, I have to quantify these cells. I need to take a lot of pictures and then quantify the cells. Ganglion cells are of 3 types and they differ in their protein expressions so I have to decide while looking at the images which cell is of which type. But in these pictures, its hard to decide.
I didn't change Tween concentration. Where did I? Yes, I do add Tween in Primary/Secondary Ab incubation. Actually I prepare my antibodies in blocking buffer as give in my protocol. I add the Donkey serum because my secondaries are made in donkey. Fluorochromes are Alexa 488 and Alexa 594.
For microscope, I only have conventional microscope. My supervisor don't want to spend money on a better microscope or let me take pictures on confocal, which is a paid service in our lab.
How can I send you my images directly through this forum?

Carl · **Carl**

Thanks very much for the details.
I hope others can offer a way to decrease background as I can't think what you can do....except for longer washes.
Re your posted image: my apologies...it was my PC that somehow corrupted the file extension.
Good image, considering it is taken witha conventional scope.
So I redownloaded it and tried a little crude PShopping
I tried to enhance the signal but only managed to increase both...I posted it in image gallery as "madsmad" .
I see your cells and their processes.
When you say "quantify" do you mean just counting number of positive cells?
After all, you can do that easily in live mode: just focus up and down, while counting with ahandheld counter?
It's far quicker, imho, than capturing images then trying to count.
You then use the confocal to take a representative image.
Are you differentiation between ganglion cell type by immunolabelling or by shape?

Re TWEEN: you permeabilise in 0.3% then use 0.05% for incubations.
imho that's far too low.
You may need to use up to 1% all the way through your immunostaining because it is very dense tissue .
I doubt that the background is due to fixation as you are keeping it quite short so, the only reason I can give for it is that the primary and secondary Abs are still trapped in the tissue matrix.
I assume that you are staining with gentle agitation?
Also, if you are doing it at 4C, don't bother: do it at RT.
Sorry I am not much help......can anyone else please suggest better approaches?

Carl

xylenefumes · **xylenefumes**

Why do the retinas from your mice liquify?

Here's a protocol from one of Reh's papers (J Neurosci. 2008 Jan 16;28(3):749-56.) in which the retinas are dissected from the eyes in cold PBS. Unfortunately, the buffer components are not listed:

Tissue preparation.
For immunofluorescence labeling, retinas were dissected in cold PBS, fixed for 2 h in 4% paraformaldehyde, rinsed in PBS, and equilibrated in 30% sucrose/PBS overnight at 4°C. Retinas were frozen (OCT, Tissue-Tek; Sakura Finetek, Torrance, CA) and cryosectioned at 12 μm. Sections were incubated overnight with primary antibodies [1:500 rabbit M-opsin, 1:500 rabbit S-opsin (both from Jack Saari, University of Washington, Seattle, WA), 1:200 rabbit S-opsin (AB5407; Chemicon International, Temecula, CA), 1:5000 rabbit TRβ2 (13), or goat NeuroD1 N-19 (SC-1084; Santa Cruz Biotechnology, Santa Cruz, CA)] and for 2 h in secondary antibodies (1:500 Alexa Fluor goat anti-rabbit 568, donkey anti-goat 488, or donkey anti-rabbit 594; Invitrogen, Carlsbad, CA). Cone outer segments were visualized with FITC-conjugated peanut lectin (L7381; Sigma-Aldrich, St. Louis, MO). Slides were viewed on a fluorescent or confocal microscope and photographed with a digital camera.

For flat-mount studies, retinas were flattened on a glass slide, fixed with 4% paraformaldehyde for 15 min, and fixed for an additional hour in a 24-well plate. Retinas were incubated in primary antibodies at the concentrations noted previously for 48 h at 4°C and in secondary antibodies overnight at 4°C. To label all cones, we incubated retinas in FITC-conjugated peanut lectin for 48 h at 4°C. Retinas were mounted in 1:1 glycerol/water between coverslips separated with an imaging Chamber (Secure-Seal; Grace Bio-Laboratories, Bend, OR). Flat mounts were viewed on a confocal microscope.

xf

arnelson · **arnelson**

To get a stronger signal you may need to increase either your primary or secondary concentration. If you have exhausted concentration solutions I would recommend trying a Perkin Elmer TSA Amplification Plus Kit and that will give you a brighter signal.

Immunohistochemistry	In Situ Hybridization	Immunostaining
Immunohistochemistry Images	Tissue staining	Pathology
Tumor Markers	IHC Reagents	Tissue Micro Array
Immunofluorescence Staining	Positive Control Slide	Polyclonal Antibody
Monoclonal Antibody	IHC Staining Protocol	In Situ Hybridization Images
Immunohistochemistry Protocol	Immunofluorescence	Immunohistochemical Staining
.................................................................................................................... .............................................................................................