I am looking for GCPRs in heart. I performed IHC and now want to quantify and report it. I am totally confused with quantification methods completely. I found some articles but they talked about reciprocal intensity, i couldnt understand what it means. Can anyone simplify and mention steps from microscopy image to final quantification.
Second, i am trying imrove quality of image by using 2010 word office ''autocorrect'' . Do you think, should i do it at the end or before analysis.
I confirmed after autocorrection result may be dangerous means it change results.
Kind reply will be highly appreciated.
Best regards,
Naseer
You can download the IHC profiler plugin and input on your image J software. Read the following for more details. its easy and fast.
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0096801
or you can just use the image J IHC toolbox to estimate the percentage of stained area and score.
https://www.dovepress.com/prophylactic-effects-of-clausena-excavata-burum-f-leaf-extract-in-etha-peer-reviewed-article-DDDT
Hello Naseer,
I would suggest you to open up the Image J tutorial and start by dragging one image into dialogue box that pops up. step wise first set invert 8 bit or 16 bit. the image turns greyscale. look for substract background. Next play with intensity keeping a minimal threshold level which should be same for all and then measure. You can look for other options to chose for but whatever parameters you chose keep them same for all the images.
I would suggest dont do much editing before the analysis else it might give erroneous results.
All the best.
Aastha.
Dear Dr. Hussein Mansour and Dr Aastha Dheer, Thank you for your great guidance. Let me try like this.
Again thanks
I agree with the comments above about using ImageJ for image processing and quantification. I suggest using FiJi that is a user friendly mod of ImageJ
Single labeling IHC quantification is all about grey scale thresholding. You will have a specific signal (Your GCPR staining) that should be "darker" than the background. Thus you define a grey threshold, below that threshold specific signal is mixed with background. Then you can measure the gray integrated density of the staining above the threshold. This is tricky when using a enzymatic reaction (like peroxidase-DAB) because small differences between samples (developing time, enzyme or substrate access etc) will result in staining variability that is not due to target concentration.
https://fiji.sc/
Hi Nasser. I am not clear on what do you exactly want to do. Measuring intensity of the binding? or extent of the binding distribution?. What type of controls are you running?. What are you testing compared to controls?. How are you processing the tissue?. Are you using whole heart slices?. I agree with the above comments but it is wise to refine your protocol and your objectives. It will facilitate interpretation of results. Good luck
You can download the IHC profiler plugin and input on your image J software. Read the following for more details. its easy and fast.
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0096801
or you can just use the image J IHC toolbox to estimate the percentage of stained area and score.
https://www.dovepress.com/prophylactic-effects-of-clausena-excavata-burum-f-leaf-extract-in-etha-peer-reviewed-article-DDDT
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