For example, immunofluorescent staining of a brain sections with NeuN/NSE (neuronal marker, Alexa 488, green color) and co-staining with calbindin D28K (Alexa 568, red color), I would like to quantify percentage of neuron positive with calbindin D28K. I am interested in actual number of cells not the intensity or integrated intensity, it is actual cell number. Does anybody has macro to run with ImageJ or some other programs, kindly share your experience. Thanks.
I simply counted the number of yellow (merge of red and green) colored cells in a field in Zeiss Laser Scanning Confocal Microscope, which has the built-in software.
Thanks Chandra. In my example I said, I wanted to quantify the number of cells with red fluorescence alone or green fluorescence alone not the merged yellow color.
When you say built in software, will it give intensity of yellow color on a 1024X1024 area or will it provide number of cells with yellow color, if so, how do you define the cells. If you can share your macro or protocol, that would help me. FYI, I use LSM710 with ZEN software. Thanks again Chandra.
Hi Venkatraman, You are welcome.
You mentioned that you would like “to quantify the percentage of neuron positive with calbindin D28K”.
Neurons positive with calbindin D28K will be yellow, as neurons stain green and calbindin D28K stain red.
I have done this work a few years ago as you could see from my 3 publications using LSM 510.
https://www.ncbi.nlm.nih.gov/pubmed/?term=somasundaram+bukoski
I do not have the protocol now, will send it to you later.
- Badges
- Science topic