Re: [Histonet] IHC for wholemount retina

From:Maria Mejia

Dr. Marcos, the following is what I did some years back on wholemount 
turle retina.
Please, keep this in mind because a number of positive changes have 
occured in
IHC techniques particularly in regard to IHC reagents.

**Use agitation in all steps.
1. transport eyecup in cold (4C) Ringer's solution.
2. fix eyecup quickly in freshly made (cold - 4C) 4% paraformaldehyde 
(PFA) pH 7.4
- 5mins.
**this period of time is sufficient for the retina to become fixed and 
firm enough to
handle. Longer fixation time will cause the retina to adhere strongly to 
the pigment
3. remove from fixative and place again in cold (4C) ringer's solution & 
using fine
forceps & scope QUICKLY, but gently tease retina from eyecup & clean 
retina of
choroid pigment. Remove as much of the vitreous & made 3-5 peripheral 
(not too big) just enough to facilitate retinal flattening & ensure even 
fixation & IHC
**complete vitreous removal is essential for good fixation & IHC labeling.
4. drop whole retina back into cold 4% PFA @ 4C - overnight (this fix 
time depends
on the antigen of interest).
5. wash retina in 0.1M PB pH 7.4 @ 4C - several changes - 1hr.
**the retina is now a free-floating thick section, but can also mount on 
slide (I did mine w/ganglion cell side up) or can place the retina FLAT 
on piece of
0.45um millipore filter for IHC.

1. treat retina in 0.5% to 1% triton/0.1M PB pH 7.4 - 1-2 hours.
2. rinse in 3 changes of 0.1M PB - 10 minutes each.
3. block endogenous peroxidase activity w/0.3% H202/(with or without)
methanol - 20mins.
**check if tissue is not wrinkled or folded.
4. wash in PBS-T x3 changes - 10 mins each.
5. second block solution using Casein super blocker (several companies 
make it, e.g.,
SkyTek, Innovex) at room temp - 20-30 mins.
6. wash in 0.1MPB x3 changes - 10 mins each.
7. primary antibody/PBS-triton/Casein.
8. wash in PBS-Triton/casein - x3 changes - 5 mins each.
9. biotinylated secondary antibody (I used a super sensitive link at 
1:50) at RT
- 1-2 hours.
10. wash in PBS-T/casein x3 changes - 5 mins each.
11. peroxidase conjugated streptavidin (I used super sensitive at 1:50) 
at RT
- 1-2 hours.
**here time will vary from 1-2 hours or longer (if longer time place in 4C)
12. wash in PBS x3 changes - 5mins each.
13. rinse in Tris buffer 50mM pH 8 - 5mins.
14. react wholemounts with chromagen solution 1.
nickel ammonium sulfate - 9.7mg
DAB - 3.7mg
50mM Tris buffer pH 8 - 25ml
microfilter solution and react - 1-2 hours
15. react in chromagen sol. 2
nickel ammonium sulfate - 9.7mg
DAB - 3.7mg
50mM Tris buffer pH8 - 25ml
250ul (0.3% H202)
microfilter and react.
**there are quite a number of chromagen methods to use this is just one 
of several
I used.
**check using scope - stop when desired intensity is reached.
16. wash in x4 changes of PB - 10 minutes each.
17. mount whole mounts on double or triple coated gel slides. Air dry at 
4 hours or overnight.
**the wholemounts on filter paper are peeled off and flat mounted on 
slides and air-dryed.
18. Coverslip.

That's it!


MariaBartola Mejia
Smith-Kettlewell Eye Research Institute
San Francisco, CA 94115

Hernan Aldana Marcos wrote:

>I need to make immunos in the whole retinas. Can anybody have a protocol to do it?
>Or a similar technique in whole embryos. Times, concentrations or any advices to make Immunohistochemistry in  thick tissues.
>Thanks in advance
>Dr. Hernán J. Aldana Marcos
>Facultad de Medicina. Universidad de Morón
>Machado 914. B1708JPD. Buenos Aires. Argentina 
>e-mail alternativo
>Histonet mailing list

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