Would you share your protocol for Duolink in brain sections?

Oligo length is 19bp

I had a macro that worked perfectly until I updated FIJI, now the “results” window will no longer open when after run(Analyze Particles…).

This is my macro, can anyone spot where the error is? Have tried asking ChapGTP and debugging line by line but I cant find anything.

dir1 = getDirectory("Please Select The Source (Input) Directory "); dir2 = getDirectory("Please Select The Destination (Output) Directory "); list = getFileList(dir1); setBatchMode(true); for (i=0; i<list.length; i++) { showProgress(i+1, list.length); filename = dir1 + list[i];

if (endsWith(filename, "tif")) { open(filename); setMinAndMax(-66, 321); run("Apply LUT"); run("8-bit"); setAutoThreshold("Otsu dark"); setThreshold(75, 255); //run("Threshold..."); run("Convert to Mask"); run("Fill Holes"); run("Set Scale...", "distance=4.4053 known=1 pixel=1 unit=um"); run ("Set Measurements...", " area perimeter shape descriptors feret's diameter Limit display add redirect=None decimal=3"); run ("Analyze Particles...", "size=5-Infinity pixel display exclude"); name = getTitle; index = lastIndexOf(name, "."); if (index!=-1) name = substring(name, 0, index); name = name + "(outlined)"; saveAs("JPEG", dir2+name); close(); } }

selectWindow(“Results”); excelname = “Resultsferet.xls”; saveAs(“Measurements”, dir2+excelname);

showMessage(“Task Completed”,“Inclusion counting is complete and the overlayed images and an Excel spreadsheet have been generated in the specified output folder”);

Hi, I need to prepare stock solutions for Collagenase I from Sigma (C0130). The provider suggests to prepare solutions in TESCA buffer (50 mM TES, 0.36 mM CaCl2, pH 7.4 at 37 degrees). I tried to dissolve TES (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, Sigma product T1375) in water but I get a very ugly grey-brownish solution. Sigma says that a "clear colorless solution at 1 M" should be obtained.

Then, when I try to adjust pH with NaOH, a brown precipitate starts to form when I add a considerable volume of NaOH (and the 7.4 pH is still far...). Have you ever prepared TESCA buffer using TES? My TES powder may be quite old...

I've also found recipes for TES buffers with Tris+EDTA+NaCl but I don't understand if it's a different buffer.

Alternatively: have you ever reconstituted the collagenase in HBSS or PBS, instead of TESCA buffer? I read that calcium is needed and maybe I may add calcium to these buffers. Thank you very much!

I have collagenase from sigma product code c9891 100mg,Type IA, 0.5-5.0 FALGPA units/mg solid, ≥125 CDU/mg solid, For general use

I do not understant from the product data sheet how many units are per mg (U/mg)....

How can I make a 30U/mL solution? How much buffer should I add to what quantity of powder to obtain 100mL at 30U/mL solution?

I would appreciate your experience. Thank you!

Hi everyone! I would like to know if I can use a different balanced salt solution instead of HBSS for reconstitute collagenase I (17100-017, Gibco), such as DPBS. I need collagenase I to dissociate skin biopsy and to isolate fibroblats from that.

Thanks!

Maria Vittoria

Hi all, I'm currently working working on a project where I use APEX2 proximity labeling to biotinylate proteins of interest. The cell lysate was first analyzed using western blotting to confirm the biotinylation reaction was successful. I then attempted to pulldown biotinylated proteins using magnetic streptavidin beads, but when running both the positive (biotinylated lysate) and negative (non-biotinylated lysate), I found no difference between samples when staining for total protein. I am aware there is some endogenous biotin in normal cell lysate, but the pulldown seemed to have no effect on reducing non-specific protein in the lysate. I used 500ug of total protein per sample to 30uL MagnaBeads and performed several stringent washes prior to elution. Does anyone have a recommendation on how to reduce non-specific binding when using whole cell lysate?

I'm measuring ADP ribose cyclase from fresh tissue homogenate using fluorescent detector ( microplane reader ). The graph shows the fluorescent of the product high at 0 mins, then decrease RFU, then at 15 min start going up so from 15 -60 min it looks like a enzyme reaction graph. Can Michealis menten equation work with this ? Cause I really don't know when the real 0 mins

Hi all.

I am doing some tests with transfections and getting this weird basal OCR profile in mitostress tests. Could someone tell me what is wrong here???

The cells look perfectly fine in wells both before and after the experiment.