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Hello everyone,

I am a radiation oncologist from Turkey and I am studying neurogenesis on hippocampus of rats. Currently my research partner in histology and I are having difficulties with staining sample tissues using CD68 (Santa Cruz), doublecortin (Santa Cruz) and Ki67 (Abcam ab16667) markers  :(

So far we have taken the steps below but unfortunately that didn't yield selective staining. Could you spot anything we possibly may have done wrong?

We also tried skipping step #8. The antibody was diluted 1/100 and we waited overnight at +4C. Most parts of the tissue were stained but there was no specific staining.

To see if the antibody is working, positive control stain was made in the intestinal sample. The antibody was diluted 1/100 and stood overnight at +4C. It stained.

After sacrification, we performed intracardiac perfusion fixation, and the tissues were embedded in paraffin within 2 weeks. We have not encountered any problems with cutting tissues with the microtome.

1. The brains of rats were embedded in paraffin after perfusion fixation procedure
2. Sections with 4μ thickness were taken
3. Deparaphinization for 1 night at incubator (at 60ᵒC)
4. Xylene for 30 minutes
5. Dehydration in graded series of alcohols (100%, 90%, 75%, dH2O)
6. Antigen retrieval for 30 min at 37ᵒC trypsin or 0.01M citrate buffer 3x1min, 3x5min (all possibilities have been tried)
7. PBS for 3x5min
8. 3% H2O2 in Methanol x5 min, 1% H2O2 in Methanol x15 min, 3% H2O2 in PBS x5 min (all possibilities have been tried)
9. Blocking solution x 5 min
10. Stained with: DCX (sc390645) diluted 1/100 and 1/50 with PBS, Ki67 (ab16667) diluted 1/100 and 1/50 with PBS, CD68 (sc7084) diluted 1/100 and 1/50 with PBS, 2 hours at room temperature, 1 hour at 37ᵒC or overnight at +4ᵒC (all possibilities have been tried)
11. PBS for 3x5 min
12. Seconder antibody for 10 min
13. PBS for 3x5 min
14.Enzyme-labeled avidin-biotin complex for 10 min
15. PBS for 3x5 min
16. DAB x 1 min, 2 min
17. Washed with dH2O
18. Hematoxylin for 1 min or no hematoxylin staining (all possibilities have been tried)
19. dH2O, 80%,90%, 100% alcohols and xylene
20. Closed with Entellan

We would be grateful if someone can point out where we possibly made a mistake? Thank you in advance.
2
Immunohistochemistry (IHC) / IHC and IF combination
« Last post by SRobert on March 01, 2017, 10:52:32 AM »
Does anyone know of a protocol for staining the same (paraffin-embedded) slide for one antigen for IHC (Vectastain ABC method) and another antigen with IF? I've been told it is possible but cannot find a protocol.  Thanks for your help!
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Biochemistry / Teduglutide
« Last post by SBella on February 28, 2017, 02:00:05 AM »
Teduglutide differs from natural GLP-2 by a single amino acid: an alanine is replaced with a glycine. This blocks breaking down of the molecule by dipeptidyl peptidase and increases its half-life from seven minutes (GLP-2) to about two hours, while retaining its biological actions. These include maintenance of the intestinal mucosa, increasing intestinal blood flow, reducing gastrointestinal motility and secretion of gastric acid.
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Biochemistry / Antide
« Last post by SBella on February 28, 2017, 01:57:45 AM »
Antide acetate (Ac-AA10-NH2) is an LHRH antagonist and represses LH and FSH release from the pituitary gland. It shows a high antiovulatory activity and releases negligible histamine.
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Immunofluorescence (IF) / Re: Staining on only the periphery of tissue
« Last post by QChong on February 13, 2017, 05:06:52 PM »
someone suggest me to cover the antibody drop with a parafilm to avoid edge effect. may be you can try. good luck.
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Immunofluorescence (IF) / unmask by proteinaseK
« Last post by QChong on February 13, 2017, 04:56:41 PM »
liver staining by CD31- rat anti mouse antibody(Abcam Catalog Number: ab28364), only the edge and around of the vessels of the liver can be stained clearly(see the attachment).
the liver to be fixed are no more than 3mm, fixed by zink fixation buffer for 72h,
proteinaseK is 20ug/ml, 30min@RT, followed by Permeabilization. 
Could you please help me to find how to stain the tissue evenly? thanks.
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I am not sure what else I can do.
The tissue is immersion-fixed in 4% pfa with a pH of 7.4 for 24 hours after perfusion.
After mounting tissue onto poly l lysine slides, I fume fix with 16% for multiple nights and then let dry for a whole day
When running CV, my tissue falls off in the very first few wells: the dH2O and 70% ethanol.

I'm at a loss as everything I find comes down to fixation quality and using coated slides.

Any other tricks out there???
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Immunohistochemistry (IHC) / Re: Mystery artifact in brain tissue!
« Last post by GAlison on February 08, 2017, 01:21:30 PM »
I've been advised to never unfreeze and refreeze tissue. I've had tissue disintegrate once I put it into the well so my solution for that was to directly mount the slice onto the slide. How are you fixing the tissue?
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Our cryoprotectant recipe calls for 50% phosphate buffer, 30% ethylene glycol, and 20% glycerol. We accidentally used phosphate buffer saline (with the ethylene glycol and glycerol) instead. I have three questions:
1) Will the NaCl interfere with cryoprotection (i.e., will it allow freezing damage to the tissue)?
2) What would freezing damage look like?
and 3) What problems could this mixup cause later with IHC?

Further details: we store our tissue at -20C and want to stain for Ki67 and doublecortin.
Thank you!
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Immunohistochemistry (IHC) / IHC artifact - aspecific staining in some tissue parts
« Last post by BCaroline on January 19, 2017, 10:26:35 AM »
Dear all,

We are facing an issue with chromogenic immunostainings on liver sections and hope you can provide some help.

We observe aspecific immunostaining in tissue regions unrelated to a particular structure (example: http://dih.irec.ucl.ac.be/dih/webViewer.php?snapshotId=14848387200379).
These stainings worked in our hands in the past and we are a bit puzzled by these results...

Those artifacts:
- are not reproducible (other regions aspecifically stained on serial sections stained different days),
- do not seem to be related to primary or secondary antibodies (same pattern for different antibodies - mouse and rabbit),
- do not seem to be caused by altered solutions (same result if using all fresh solutions).

Any advice welcome  :) :)

Thank you in advance,
Caroline



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