Search found 11 matches

by douglas.linn
Mon Feb 04, 2019 7:22 pm
Forum: Reagents
Topic: Standardized retrieval conditions?
Replies: 1
Views: 503

Re: Standardized retrieval conditions?

Hi Santhosh, I haven't tried anything beyond the recommended EDTA buffer for 10 minutes at 95C. I'm using the Biogenex microwave. I have stuck with these conditions mostly because those conditions are similar to what most antibody manufacturers recommend for IHC, at least for the antibodies I'm usin...
by douglas.linn
Tue Jan 29, 2019 9:04 pm
Forum: Reagents
Topic: Detecting mouse monocytes on FFPE?
Replies: 0
Views: 469

Detecting mouse monocytes on FFPE?

Hi All,
Has anyone has success detecting mouse monocytes using mouse FFPE tissues? Using IHC, I can readily use Ly6C (ER-MP20) or CD115 (sigma polyclonal), but neither of these antibodies adapt well to IMC. Does anyone have any other clones that work in their hands? Thanks!
-Doug
by douglas.linn
Fri Oct 19, 2018 3:17 am
Forum: Data analysis
Topic: Cell population frequencies in HistoCat
Replies: 10
Views: 2874

Re: Cell population frequencies in HistoCat

Does this issue address your question: https://github.com/BodenmillerGroup/histoCAT/issues/11 It works, but I have to manually count the number of rows from each respective ImageID/ROI. This would become quite tedious when there is a lot of clusters across a large set of ROIs, so I was hoping those...
by douglas.linn
Mon Oct 01, 2018 2:20 pm
Forum: Data analysis
Topic: Cell population frequencies in HistoCat
Replies: 10
Views: 2874

Re: Cell population frequencies in HistoCat

Denis, thanks for adding the cell number function in the latest version of Histocat! Is there a way to determine the contribution of each individual ROI/image to those numbers? Perhaps I missed it, but plotting ImageId vs Amount_cells_in_sample doesn't seem to work
by douglas.linn
Tue Sep 18, 2018 5:26 pm
Forum: Reagents
Topic: Mouse FFPE antibodies
Replies: 5
Views: 1702

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: 2883

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: 2883

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: 1969

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: 2919

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: 1528

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).