Trypsin HCl BrdU

dcwebste · **dcwebste**

Hi I'm really new at this and have been reading papers and protocols for days to no avail.

I want to do fluorescent BrdU immuno. The ultimate goal is to do double immuno with BrdU and an antibody for a gene of interest.

I have BrdU labeled brains that have been prepped for paraffin sections, cryo sections and vibratome sections since I don't know what will work.

QUESTION
Where do you purchase trypsin (cat number), how do you prep, store, use etc? People in my lab are buying a Invitrogen kit and NOT using the antibody just the trypsin which is ridiculous.

HCl, what conc do you use at what temp and how long? I assume this is what the Invitrogen kit "denaturing solution" is replacing.

Any other advice or protocols even to start with would be great.

We have an Invitrogen kit (93-3943) so this antibody as well as Dako M0744 and a freezer log says we have "Abcam sheep BrdU" which may or may not be true.

Also any references, books, etc. I've never done fluoro immuno and did very little DAB based and I've never done double immuno so I may use things that are incompatible etc. I'm just feeling a lot lost.

Carl · **Carl**

....
Yes, a tall order to sort all that out.
Why not have a look in the IHC Protocols module here..all answers re BrdU disclosure there.
I don't use trypsin/HCL protocol anymore but rely upon Citric acid HIER and one partic Ab, that works in Pwax sections after HIER.
I have tried several other Abs and none work.
That is NOT to say that they will not work, after HIER , in other Labs.
Other labs swear by the trypsin /HCL method.
Some labs, using perfuse-fixed frozen sections, use HCL alone, with great success. ( makes sense as such fixation is minimal and there's no alcohol/heat involved....from the Pwax processing, so no need for digestion)
You just have to spend a week or so going thro the variables and come back to ask, if neccessary, about specific points.
So, good luck.
Sure, keep firing away and we will try to help.
carl

liver · **liver**

This is my first time on this forum, so bare with me.

I have been doing BRDU for a long time. I have always used a 2N HCL for one hour at room temp.

But first you need to do antigen retrieval (citrate, I use Dako's) for 25 min. then follow with the 2N HCL.

Hope this helps.

Carl · **Carl**

It is an interesting variation.
Please tell us which anti BrdU antibody you use.

Thanks.
Carl

liver · **liver**

The antibody I use: Rat MAB Anti-BRDU, cat#0BT0030G
I have been using it on my mouse tissue.

Before I found that Ab. I was using Dako's BRDU with their ARK kit for mouse on mouse. The rat antibody is very good.

Angela

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
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