Search found 17 matches

by douglas.linn
Tue Sep 18, 2018 5:26 pm
Forum: Reagents
Topic: Mouse FFPE antibodies
Replies: 5
Views: 2495

Re: Mouse FFPE antibodies

Matt,
In my hands CD19 (6D5) doesn't work for FFPE mouse tissue, but does for frozen. CD8 (4SM15) works fine for me though. The other clones you mentioned I haven't tested but overall I tend to prefer abcam & CST clones for mousef-reactive FFPE antibodies.
-Doug
by douglas.linn
Wed Jun 20, 2018 2:42 pm
Forum: Reagents
Topic: Nuclear/DNA staining with frozen sections (OCT embedded)
Replies: 5
Views: 3957

Re: Nuclear/DNA staining with frozen sections (OCT embedded)

Yes, in my hands acetone or methanol fixation gives better resolution of surface markers than PFA, for example for CD8a clone 53-6.7. PFA fixation is still acceptable and may be fine for downstream image analysis. I have found that 70-80% of antibodies ( whether human and mouse-reactive) that we rou...
by douglas.linn
Fri May 25, 2018 6:10 pm
Forum: Reagents
Topic: Nuclear/DNA staining with frozen sections (OCT embedded)
Replies: 5
Views: 3957

Re: Nuclear/DNA staining with frozen sections (OCT embedded)

Santhosh, Thanks for the suggestions. I've tried a few concentrations and incubation timings like you suggested but to no avail in either acetone or methanol fixed cryosections. For PFA-fixed cryosections the 191/193 works totally fine (perhaps that is what you were using?), but in my experience doe...
by douglas.linn
Fri May 25, 2018 6:06 pm
Forum: General
Topic: IMC staining - blocking
Replies: 4
Views: 3020

Re: IMC staining - blocking

I would be very surprised if Fc blockade was necessary for IMC as it is not in most standard IHC assay protocols. In fact, even though the serum (or BSA) blocking is traditionally thought to block FcR during IHC/IF/IMC, an interesting paper found no nonspecific staining when omitting the blocking st...
by douglas.linn
Fri Feb 09, 2018 8:36 pm
Forum: General
Topic: Best instrument for heat retreaval in FFPE
Replies: 7
Views: 4104

Re: Best instrument for heat retreaval in FFPE

We use the Biogenex EZ Retriever system which Jared Burks highly recommended at the users meeting
http://store.biogenex.com/us/ez-retriever
It is expensive, but maintains a very consistent temperature without risk of buffer boiling over like a standard microwave.
by douglas.linn
Tue Jan 30, 2018 8:48 pm
Forum: Reagents
Topic: Antibodies for FFPE
Replies: 2
Views: 2044

Re: Antibodies for FFPE

I would concur that many CST antibodies work quite well, especially for mouse FFPE tissue. I also have had good success with abcam's "IHC-P" validated antibodies, particularly the EPR clones (rabbit monoclonal).
by douglas.linn
Mon Jan 22, 2018 7:00 pm
Forum: Reagents
Topic: Nuclear/DNA staining with frozen sections (OCT embedded)
Replies: 5
Views: 3957

Nuclear/DNA staining with frozen sections (OCT embedded)

I'm just curious what others are using to broadly stain nuclei/DNA when working with OCT-embedded frozen samples. Most standard IHC/IF protocols utilize acetone or methanol, but these seem to destroy quality of nuclear proteins for IMC, so 191/193 Ir and Histone-H3 (clone D1H2) don't stain well (whe...