We published a simple method (using Adobe Photoshop and Image J) to immunosignal quantification:
"Application of Photoshop and Scion Image analysis to quantification of signals in histochemistry, immunocytochemistry and hybridocytochemistry" .
It is easy, quick and cheap. Try it. Good luck
In cellular pathology Ki-67 proliferation index is used. Which is sort of semiquantitative assessment of average percentage of Ki-67 positive cells in a given tissue/lesion/tumour. I hope this is of some help to you.
Use either a morphometric software (I reckon there must be also free ones) or an online service (I used the one from the TUM spin-off - I can't remember the name exactly). The cheap alternative is "eyeballing" which involves making a rough estimate (based on previous experience), or counting the number of +ve cells compared to the total number of nuclei in a field. All these approaches are sensitive to thresholding for staining intensity, which is rather subjective and also depends on pre-analytica factors.
I hope this helped.
Alberto
I use image J and manually count 1000 cells of interest per animal (n). Our group hasn't had any luck with software that detect nuclei or automatically counts.
Hello,
I used ImageJ as well, you'll need a plugin named "Colour Deconvolution" and change your image to 8bit, it differentiates the H DAB from Ki67 stained cells although you need to play a bit with threshold and sharpness of the image, its 95% accurate.. I am attaching a file explaining the process in a flowchart hope it helps!!
All the best!!
We published a simple method (using Adobe Photoshop and Image J) to immunosignal quantification:
"Application of Photoshop and Scion Image analysis to quantification of signals in histochemistry, immunocytochemistry and hybridocytochemistry" .
It is easy, quick and cheap. Try it. Good luck
We have counted Ki67 labelling using ImageJ, considering a percentage of positive celles in five different fields of each case. We assign different values depending on the percentage of positive cells: 0= no positive cells, 1= 1 to 25 % of positive cells, 2= 26 to 50 %, 3= more than 51 %.
Simple method with eye piece you count the labeled cells in selected lenght
You might try FIJI, whcih is associated with Image J and a jazzed up version. Sharon Vinsant
We also use ImageJ with good results.
Attached you find one of our articles about how to set up ImageJ.
You can use either colour deconvolution (as mentioned by Falguni Patel) or, as we use it, Colour Threshold.
I agree with the majority of posts here and say define a size for your region of interest then count the total number of cells per high power field and as a percentage.
One thing to consider with IHC is to determine a threshold of staining. So come up with a rule that you will use for every high power field. For example "If I have to think about whether that cell is positive, I will not include it in my count"
So after quantification I will usually have a couple of results:
1. Strongly positive cells
2. All positive cells.
Positive immunostaining of basal cell layer and some cells of the Prickel cell Layer which are dividing cells. the area percent is calculated by using image analyzing software.
Dr nadia
Thank you for all these information, it's really cool to know those amazing plugins for imageJ and to use the percentage to assess the grade. I'm still searching for the optimal threshold...
Hi, we normally use the software ImageAccess, which has an particel analysis module to count all cells and Ki-67 positive cells. This particale modulce has special filter options to optimize the detection. Additionally, we could calculate the amount of ki-67 positive cells to the investigated area (mm2).
I count two different areas with 200 cells in each and give the percentage of positive cells. In a sample like the above picture the basal cells are the only ones expected to proliferate. The Ki-67 looks very normal to me. So the stained cells should be 95-100%= normal. If you find positive cells in the third upper layer of the epidermis that is not normal and should be reported separately.
Just showing positive expression of Basal cells, not very special, I think.
Hi,
I recommend using an eye grid and count number of positive cells using the 40x objective.
Sverre H. Torp
hi,
can anyone pls explain scoring calcualtion too.i have manually counted Ki-67 cells in 5 fields as 60,45,60,57 and 55 ,what do i do next ?
You can use the on-line software ImmunoRatio, from University of Tampere. ImmunoRatio is a free web application for automated image analysis of estrogen receptor (ER), progesterone receptor (PR), and Ki-67 immunostained tissue sections.
The web address is: http://153.1.200.58:8080/immunoratio/
Tuominen VJ, Ruotoistenmäki S, Viitanen A, Jumppanen M, Isola J. ImmunoRatio:
a publicly available web application for quantitative image analysis of estrogen
receptor (ER), progesterone receptor (PR), and Ki-67. Breast Cancer Res.
2010;12(4):R56
Hi Tibor Mezei your link about the quantification is helpful. Can you please say how many images one should quantify ? is it 5 different fields in a slide. then should it be averaged?
Hi Patrick
I am trying doing Ki67 IHC in lungs, But not successful. Can you please help me with sharing a protocol?
- Badges
- Science method