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Author Topic: Somatostatin IHC help  (Read 1624 times)

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Offline SDashiell

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Somatostatin IHC help
« on: March 05, 2018, 11:42:19 AM »
Has anyone successfully done an immunohistochemical stain against somatostatin? If so, could you kindly share your protocol, including specific primary and secondary antibodies?

Our lab has tried numerous protocol variations to no avail on 40 um mouse brain sections--we either get no signal or very patchy, light cell body staining near the outer edges of the sections. We have been using Millipore MAB354 primary and Invitrogen A11006 secondary.

If anyone could share some insight on staining this pesky protein, it would be very much appreciated!

Below is our latest IHC protocol:

3 x 20 min. wash in PBST
2 h blocking step
48 h Primary incubation at 4C (1:200 in PBST)
3 x 20 min. wash in PBST
4 h secondary incubation (1:500 in PBST)
3 x 20 min. wash in PBS


PBST:
0.1% Triton X-100
in 1x PBS

Blocking solution:
0.1% Triton X-100
5% normal goat serum
0.2% bovine serum albumin
in 1x PBS

Thanks very much!

-Dash

Somatostatin IHC help
« on: March 05, 2018, 11:42:19 AM »

Offline RSabine18

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Re: Somatostatin IHC help
« Reply #1 on: December 27, 2018, 10:46:25 AM »
This is a late answer, but...

We are currently using a protocol to visualise somatostatin with immunofluorescence on 50 m-thick free-floating mouse brain sections, and it works fairly well.
Primary antibody : polyclonal, made in goat, D-20 from Clinisciences, used 1/500 (overnight at 4C)
Secondary antibody  : Donkey anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, DyLight 488, used 1/1000 (3h at room temperature).
-   Washing buffer : PBS + 1% Normal Donkey Serum + 1% gelatin from cold water fish skin
-   Blocking Solution : PBS + 5%  Normal Donkey Serum + 2% gelatin from cold water fish skin + 0.2% TritonX-100
-   Diluting buffer for antibodies : PBS + 1% Normal Donkey Serum+ 1% gelatin from cold water fish skin + 1% DMSO + 0.2% TritonX-100

I hope it may help you.
Good luck,

Sabine

Re: Somatostatin IHC help
« Reply #1 on: December 27, 2018, 10:46:25 AM »