I am performing IHC on skin tissue to investigate a group of novel cells. I have included iso controls for all of the different stains I have done, but am still having trouble distinguishing low expression from no expression. I am also trying to separate some of the expression into high, intermediate and low. Does anyone have any suggestions for standardising this sort of thing, or perhaps could suggest a computer program which may be able to help me with this? Thanks.
Thanks so much for the advice! Have found it super helpful! :D
Try a special plugin, compatible with ImageJ - IHC Profiler. It has a lot of different features, hope it will help you.
http://sourceforge.net/projects/ihcprofiler/
For a preliminary analysis you could use the function "histogram" of Photoshop (you find it in "Window"), but you need to select the area occupied by cells before. In this way you can obtain a measure of average level of signal in one particular channel (blue-grey-red) per single cell (if you select them one by one), or per each population (if you select them all). Of course you need high quality photos and a negative control (no signal).
hope it helps!
Carmen
There are quite a lot of different analysis module in ImageJ (which is freeware).
I've done a bit of IHC (in lung) and it is fairly problematical to link it to expression level (mainly due to the fact that it is hard to distinguish between a low signal caused by low target and low signal caused by poor antibody binding).
I think to convince people that you are measuring all the protein that is there you would have to have at least two different antibodies for each target (to two different epitopes of the same protein) and get the same signal intensity in your section for both. Also (ideally) a positive control for the antibody (although this might not be possible).
If you do get no signal you might also want to homogenise & lyse the tissue and run a western blot and probe with the antibody (to show that there is low/no expression). This is only possible however if the ab does not bind to other cells/structures in your tissue.
Then off course there is the issue of what type of antigen retrieval you did (as some ab's prefer trypsin mediated, some Citrate etc). One thing I found that improved the signal more than anything else was to include 0.5% Triton as a "penetration enhancer" during the primary incubation step (it's high I know but this was a tip from IHC World and it worked well form me).
I have assumed this tissue is PFA fixed, paraffin wax embedded tissue - if it is frozen then the penetration enhancer probably won't make much difference.
Sorry I can't be more help,
Gary
Hi Megan,
That is what I do when i have no idea about IHC scoring patterns like ASCO/DAKO-Her2, Allred etc
Just make a simple scoring system. When there is no guideline for scoring I do the following:
I plot area and intensity:
Area in percentage 10-30%< - 2, >30-50%< - 3, >50-80%< - 4, >80%-5
Intensity: 0(no immune reactivity), 1 (detectable but faint or patchy in the cell), 2 (weak homogeneous or moderate irregular), 3 (moderate homogeneous or strong patchy), 4 (strong homogeneous)
Then plot area by intensity and sum the different results
1(area) x 4(intensity) + 2(area)x3(intensity) +4(area)x0(intensity)=10
It works for me :)
Cheers and good luck!
Ivan
Make sure that your image is 8 bit, so it is in the 0-255 pixel intensity. Then use ImageJ to find the adequate thresholds for background, low intensity, medium intensity and high intensity. Use the same thresholds for all your images, provided that you have acquired them using exactly the same parameters (exposure for normal microsopy, PMA gain for confocal, same laser power, same objective, etc). ImageJ will let you do different kinds of measurements in the pixels between each pair of thresholds (average intensity, number of pixels, integrated intensity, etc).
If what you want is to increase the intensity of a low intensity fluorescence label, consider using tyramide signal amplification (TSA).
Try a special plugin, compatible with ImageJ - IHC Profiler. It has a lot of different features, hope it will help you.
http://sourceforge.net/projects/ihcprofiler/
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