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trouble with IHC


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#1 Vu Hai Ha

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Posted 28 June 2016 - 08:50 PM

Dear 

Recently, I have some troubleshooting with IHC experiments. If anyone has experience about IHC, please help me solve this problem

I try to detect macrophage cells in mouse brain tumor sample with CD68 antibody (CD68, Cat # MCA1957, serotec, rat anti-mouse). The company report that this antibody can be used for paraffin section. howerver, I already tried several times but i could not get results in my sample (paraffin section).

I tried to optimized primary antibody dilution from 1:1000 to 1:20, but all those processes were failed.

I used antigen retrieval method by microwave heating with microwave

Please help me

Thank so much. 



#2 bob1

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Posted 30 June 2016 - 05:48 AM

Did you try the protocols and references suggested by the webpage for that antibody?



#3 mdfenko

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Posted 30 June 2016 - 05:55 AM

would you give us your protocol, including paraffin removal?


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#4 merlav

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Posted 30 June 2016 - 12:14 PM

also the incubation time and if possible one photo. Do you have a positive control? 


Science without religion is lame, religion without science is blind.
Albert Einstein

I am among those who think that science has great beauty. A scientist in his laboratory is not only a technician: he is also a child placed before natural phenomena which impress him like a fairy tale.
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#5 Vu Hai Ha

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Posted 30 June 2016 - 08:25 PM

This is my protocol for IHC with that antibody

1. warm slice at 60 degree in 1hr

2. deparraffiniza in zylene 3 times (3 munutes/1time)

3. Rehydate (from 100%EtOh-95%Etoh-90%EtOh-80%Etoh-70%Etoh, 3 minutes/1 step)

4 washed in ruuning H2O

5 antigen retrival: microwave (100 degree 5 minutes, 95 Degree in 15 minutes)  in 10mM sodium citrate buffer pH6.0

6. Cooling in RT 30 minutes

7. washed with 1xPBS, 3 times

8. incubate 0.3%H2O2, 30 minutes, RT

9. wash 3 times with PBST

10. Blocking: normal goat serum with tritonX-100 (0.1%)

11 primary antibody: with series dilution: 1:1000 to 1:20, incubate overnight, and the next day, 6hrs in RT

12. washed with PBST, 3 times, 10 minutes/1 time

13. Secondary: goat anti-rat IgG biotin conjugated, 1:250, 3hrs in RT

14.wash again 3 times

15. Abidin-biotin complex, 1hrs in RT

16. wash again

17. DAB reaction in 50mM tris-Hcl, 0.5 mg/ml diaminobenzimide, 0.03% H2O2

 

I already searched some reference in datasheet of that antibody, I found that all the reference only use for frozen section. However, that company reported that they could detect signal in parraffin section. That why, I got many troubleshooting with that antibody.

Please give me some recommendation for this issue



#6 mdfenko

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Posted 01 July 2016 - 04:08 AM

how did you control the temperature in the microwave?

 

are you sure you weren't supposed to use a hot plate or some other type of oven?


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#7 merlav

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Posted 01 July 2016 - 09:48 AM

1. warm slice at 60 degree in 1hr-ok, also could be left overnight

2. deparraffiniza in zylene 3 times (3 munutes/1time)-are you making sure that all paraffin is completely  eliminate?I usually deparaffinized for at least 8 min/ea and use no less than 2 bath of xylene, check slides.

3. Rehydate (from 100%EtOh-95%Etoh-90%EtOh-80%Etoh-70%Etoh, 3 minutes/1 step)

4 washed in ruuning H2O- I place slides in dH20 for 30 seconds

5 antigen retrival: microwave (100 degree 5 minutes, 95 Degree in 15 minutes)  in 10mM sodium citrate buffer pH6.0-as told mdfenko you can't control microwave temp it is High, medium, low.  I had use a pressure cooker for microwave (household from amazon) 5 min at high temp then 10-20 in low temp, water bath set at 95C and a steamer (household also) what I found that worked better was the pressure cooker it cost less than $50

6. Cooling in RT 30 minutes​-check if that antigen retrieval needs water to stop the reaction once its has cool down and before washing.

7. washed with 1xPBS, 3 times

8. incubate 0.3%H2O2, 30 minutes, RT the H2O2 is prepare with MetOH? what H2O2 you use? the one of the drugstore? (it is 3% so have to take in account that) or 30% from a chemical company? in brief check if you have the correct percent.

9. wash 3 times with PBST

10. Blocking: normal goat serum with tritonX-100 (0.1%)

11 primary antibody: with series dilution: 1:1000 to 1:20, incubate overnight, and the next day, 6hrs in RT- make sure you use a humidity chamber that is for slides, to make sure it is leveled. 4C overnight should be more than enough.Also use a PAP Pen to keep solutions covering the tissue sections.  usually for IHC 1:200 to 1:50 is needed. A dil of 1:1000 is usually use in Western Blot

12. washed with PBST, 3 times, 10 minutes/1 time

13. Secondary: goat anti-rat IgG biotin conjugated, 1:250, 3hrs in RT

14.wash again 3 times

15. Abidin-biotin complex, 1hrs in RT

16. wash again

17. DAB reaction in 50mM tris-Hcl, 0.5 mg/ml diaminobenzimide, 0.03% H2O2

Did you use a counterstain as hematoxylin ?

Hope this help, if not then my advice is to change the antibody.  


Edited by merlav, 01 July 2016 - 10:39 AM.

Science without religion is lame, religion without science is blind.
Albert Einstein

I am among those who think that science has great beauty. A scientist in his laboratory is not only a technician: he is also a child placed before natural phenomena which impress him like a fairy tale.
Marie Curie

#8 Vu Hai Ha

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Posted 05 July 2016 - 04:55 AM

Thank so much for all your suggestion. I tried several steps in my protocol as being mentioned above. Fortunately, I already got positive signal with my sample. 

Again, thank all of you.






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