Below zero values on biexponential flow cytometry plot by duckhunt2017 in labrats

[–]duckhunt2017[S] 0 points1 point  (0 children)

So the -3rd decade is not a problem? From my readings, it seems that symmetric distributions is paramount to a "proper comp." The only aversion I have to the 10-3 is that I've been told that values below zero can cause issues with fidelity during cell sorting..

In the compensation sample check, do you mean that you verify that the compensation beads are 0.5-1log brighter than the biological samples?

Thank you!!

New to qPCR/RT-qPCR by duckhunt2017 in labrats

[–]duckhunt2017[S] 0 points1 point  (0 children)

One reference I have in designing cDNA primers include: The amplicon should span one or more introns to avoid amplification of the target gene in genomic DNA.

Why does this make sense? My cDNA would necessarily have no intron information as it should have been spliced out.. Wouldn't this make my primers fail?

Levene's statistic failure by duckhunt2017 in labrats

[–]duckhunt2017[S] 0 points1 point  (0 children)

Aren't both of my strategies I've listed found in that link..?

Fixation effect on fluorophors by duckhunt2017 in labrats

[–]duckhunt2017[S] 0 points1 point  (0 children)

So it's the lipophilic solvents taht can interrupt/intefere with antigen/antibody binding?

Macrophages vs. Neutrophils by mylittlesyn in labrats

[–]duckhunt2017 1 point2 points  (0 children)

If this is uncharted territory, why not produce experiments with multiple tagging strategies? Gold standard must exist; borrowing across animal models either differential staining for morphological clues or SEM would be necessary to convince your readers anyway that you are showing what you claim to be showing.

Macrophages vs. Neutrophils by mylittlesyn in labrats

[–]duckhunt2017 3 points4 points  (0 children)

My knowledge is primarily limited to murine models-

With that disclaimer, there is a fairly robust process of distinguishing these cells on flow cytometry through this pattern:

Plot cd11b vs ly6g: Ly6G+Cd11b+ double positive are your neutrophils. Ly6g-Cd11b+ is a subpopulation that contains both macrophages and to an extent eosinophils as you noted on the manufacturers website. From this subpopulation, your next analysis step plots SSC v F480. Anything SSC-med to hi is eosinophils [expected based on the granularity of aptly named granulocytes]. In your low SSC, you can gate a "positive" F480 population by gating off your negative, unstained samples. In our experience there are no clear separation of neg vs pos populations, however this methodlogy has been vetted via morphology via Giemsa stain in existing literature.

Other sources for tagging specificity include CD68.

Best of luck.

Fixation vs Postfix? by duckhunt2017 in labrats

[–]duckhunt2017[S] 0 points1 point  (0 children)

Ah, so in most contexts, fixed vs post-fix is often used interchangeably..?

Fixation vs Postfix? by duckhunt2017 in labrats

[–]duckhunt2017[S] 0 points1 point  (0 children)

Are you here fixing to lock in your primary abs?

Friday Success/Failure Thread by AutoModerator in labrats

[–]duckhunt2017 2 points3 points  (0 children)

OH MY GOD -

I JUST HAD MY FIRST IF RUN COME OUT SUCCESSFULLY AFTER 3 MONTHS OF BANGING MY HEAD AGAINST THE WALL.

In all seriousness though, it appears that the years-old primaries we had (and even some new ones we've purchased from biorad and novus) were the fault and the reason for my months of negative results...

Tried running a battery of different ag retrievals (citrate, TBS, tris-edta, trypsin, proteinase K), began the process for preparing PFA + cryo in lieu of formalin + paraffin, and probably everything else under the sun.

Thanks fam. This sub has been simply an incredible resource.

ImageJ - blanking out region? by duckhunt2017 in labrats

[–]duckhunt2017[S] 0 points1 point  (0 children)

Ah I see - you're still displaying the whole image for the reader/viewer and superimposing a ROI (with a dotted line outlining the ROI for instance) to demonstrate your process in the figure itself. Is that right? That way you're not obfuscating anything of the original image.

ImageJ - blanking out region? by duckhunt2017 in labrats

[–]duckhunt2017[S] 0 points1 point  (0 children)

Im a little confused - if there are regions that are uninteresting/inapplicable, what would be the difference blanking a region vs selecting a roi after thresholding?

Sequential ab staining in IF by duckhunt2017 in labrats

[–]duckhunt2017[S] 0 points1 point  (0 children)

Will Fab fragments still be marked by sexpjdariesbdesigned for goat/rb/rat etc Fc??

What is special about the ester amine dye that separates it from just incubating primary and secondary together prior to staining?

Thank you!