Search found 13 matches

by douglas.linn
Fri Aug 02, 2019 6:50 pm
Forum: General
Topic: Planning/Calculating Runtime & Experimental Details
Replies: 2
Views: 412

Re: Planning/Calculating Runtime & Experimental Details

In my experience, acquiring at 200Hz takes a little under 2 hrs per 1mm^2
by douglas.linn
Fri Aug 02, 2019 1:54 pm
Forum: General
Topic: length of time after sectioning
Replies: 2
Views: 180

Re: length of time after sectioning

We try to stick with as fresh as possible but have gotten fine results with slides 6+ months old. My impression is that it depends on how the cut sections are stored, how abundant/stable your epitope is, and how good your antibody is. At the 1st IMC UGM, there was a discussion (David Rimm I believe)...
by douglas.linn
Mon Feb 04, 2019 7:22 pm
Forum: Reagents
Topic: Standardized retrieval conditions?
Replies: 1
Views: 628

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: 929

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: 12
Views: 3332

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: 12
Views: 3332

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: 1898

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: 3126

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: 3126

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: 2238

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...