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21
Neuroscience / Somatostatin IHC help
« Last post by SDashiell 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
22
Immunohistochemistry (IHC) / Unspecific staining in skin
« Last post by DValerie on February 23, 2018, 01:45:07 PM »
Hi!

I am doing IHC in paraffin-embedded tissues and I have problems with a unspecific staining. I work with equine wounds and I know that unspecific staining has been seen in sebaceous glands, but I have one also in the blood clot. I made a test to check if this was probably caused by the endogen avidin/biotin and I got a positive staining even without primary and secondary antibodies (I use ABC with alkaline phosphatase). So, do you guys think I should block endogen avidin/biotin or this may due to something else?

Thank you!

23
Immunohistochemistry (IHC) / Fc Fusion proteins used as IHC primary antibody
« Last post by CSuzanne on February 13, 2018, 11:32:49 AM »
Hi guys,

I am reviewing data from a CRO attempting to develop an IHC assay for us using a fusion protein and trying to detect its receptor via a standard IHC frozen tissue method with brief acetone fixation.  I don't believe it is working and am trying to find some literature on using Fc fusion proteins in this manner to better understand what conditions one needs to use, sensitivity of the assay, etc.   Does anyone have experience with this?

Thanks, Suzanne
24
Antibody C / Cyclin D1 Monoclonal Antibody (DCS-6)
« Last post by ZRossella on February 01, 2018, 09:58:53 AM »
Does anybody have any experience with Cyclin D1 Monoclonal Antibody (DCS-6) on Benchmark GX with ALK PHOS RED DETECTION KIT?
I've tried 1:50 and 1:100 diluition, CC from 0 to 30', with/without amplification and antibody diluent with/without casein block... slides going from a big cherry to a like-white tissue... making me crazy...

thanks in advance
25
I did an ICC and while imaging, I found that there are salt crystals on the coverslips. How can I avoid it?

My last steps are as follows:
After secondary, I wash the coverslips with PBS 3 times for 5 min.

I put a drop of my mounting media on a glass slide.

Take out the coverslip from the PBS, gently tap it on a kim wipe and place it on the glass slide.

Next, I keep the slides overnight at RT in dark and the following day I seal them using nail varnish.

Thanks
26
General Histology / Oil Red O mysterious precipitate
« Last post by JRodrigues on January 05, 2018, 10:30:44 AM »
Hi everyone,

I'm staining some lipids in cell cultures and I found these weird precipitates (or something else) that I haven't found before. I've attached a picture.

Do you know what it can be?

Thanks for the help.:)

Best,

Joana
27
Miscellaneous / tissue handling post cutting
« Last post by JHofmeister on October 16, 2017, 10:23:22 AM »
Half rat brain tissue pfa fixed, frozen cut on cryostat 30microns stored in cryoprotectant.
Brain curled tight while stored and tears easily when trying to uncurl for mounting on gel slides after staining.
any suggestions would be helpful.
28
Antibodies Review / EGR1 antibodies
« Last post by Histolab on September 12, 2017, 05:22:09 AM »
Hi,

Can anyone recommend a good Polyclonal EGR1 antibody as a replacement to the SC-189 that has been discontinued by santa cruz,

thank you in advance
29
Immunofluorescence (IF) / Tdtomato fluorescence disappears in OCT frozen sections
« Last post by PEpstein on August 18, 2017, 01:16:21 AM »
I have many frozen tissue tissue blocks containing a cell specific Tdtomato marker.  If the sample was formalin fixed sections retain great tdtomato flourescence. But for samples not formalin fixed sections lose tdtomato flourescence immediately on exposure to any aqueous buffer.  Does anyone know why this happens and how we can prevent loss of tdtomato fluorescence in our OCT non-formalin fixed samples?
30
Immunohistochemistry (IHC) / Staining for acidic pH in formalin fixed tissue
« Last post by JMeehan on August 03, 2017, 11:57:14 AM »
Does anyone know of a way to stain for acidic pH areas in sections cut from tissue that has already been formalin-fixed and embedded in paraffin? I know there are methods to assess pH in tissue that has not yet been formalin-fixed, but I am not aware of any that can be used to give an idea of the pH in different areas after the tissue has been fixed. Any help would be greatly appreciated. Thanks, James.
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