You would have to share your protocol to be able to provide you with an insightful answer to your question. But to begin with, the predominate estrogen receptor subtype in the ovary is EsR2 and in the endometrium it is EsR1. Thus it may not be surprising the you are not seeing much in the way of positive staining for EsR1 in the ovarian granulosa cells. Then there is the question of the antibody dilution that you are using and what are you using for a chromagen? If you are using DAB you may need to quench the endogenous peroxidase activity in the ovary before you begin IHC. You could also switch to using a fluorescent method which could cut down your back ground staining as well. Good luck to you.

thank you Mr. Warren for your help

1.after deparaffinization and hydrate, we done Ag unmasking (immerse the tissue in citrate buffer) in microwave for 15 minutes, incubate the slide in 3% H2O2 in methanol for 15 minutes, wash in PBS, incubate with normal horse serum (30 m, 37 0C), wash in PBS, incubate with primer AB (overnight 4 oC), wash in PBS, incubate with second Ab (30 m, 37 oC), incubate with ABC kit (vector lab) for 30 m 37 OC, wash in PBS, incubate with DAB, then counterstain

we use primer Ab for EsRalpha that ready to use from Dako. we use about 1-2 drops

2. We use DAB for a chromagen and we have done to block the endogenous peroxidase activity with 3 % H2O2 in methanol for 15-30 minutes

3. for florescent method, we dont have the chromogen that labeled flourescent

to detect receptor, what is the critical step that we have to give more attention in IHC

Which kind of negative control do you use? tissue without the antigen or same tissue as test-specimen with same protocol without primary?

If you use different tissues, it may be a matter of fixation. Estrogen receptor is susceptible for ethanol-fixation, as far as I remember.

What is your fixation-protocol?

thank you Ms Gundrun

we use the same tissue as test -specimen with same protocol without primary Ab.

For fixation, we use  4 % paraformaldehyde solution for 2 days

Have you performed other IHC tests with this procedure successfully? Are the species of your protocol compatible?

primary is from mouse, secondary is from which species against mouse?

I assume, that incubation over night with the ready-to-use antibody is too long. Ready-to-use needs usually about 30 min incbation at RT. This could cause unspecific staining in the negative control.

Can you exclude, that there happend no mistake with the application of primary and negative control?

I would try to repeat the test with either diluted primary or shortened incubation, until the negative control is clear. A well-known positive control should also be stained with the experiment (breast, portio).

thank you for your advice Ms Gudrun

i have done IHC procedure to detect ir-FSH cells on the pituitary gland of rat and the result was successfully. But in the ovary, i have tried more than 5  times but the result was not successfully.

i will try to incubate primary Ab about 30 min at RT

if you don't mind, can i get the IHC protocol that you usually do in your lab.

thank you

We do usually automated IHC - so this protocol would not help you very much.

In your situation I would doubt, if the antibody does the correct job.

thank you Ms Gudrun

would you mind to give advice for us to running the manual method of IHC in our lab ?

I would recommend biotin-free methods like polymer-methods. This is, how I would start.

1. Cut paraffin sections 3 µm, adhesive slides,  let dry vertically to get rid of any water under the section. (no delay of staining more than a few days).

Bake 30 min at 60°C (no higher dry heat)

2. Deparaffination in xylen (or similar) 2x 5 min, with agitation.

3. Rehydration through ethanols 96-70-50 each 2 min with agitation, til water

4. Antigen-Retrieval. (like you have done) 15 min cooking, 15 min cooling at RT

EDTA-Tris-buffer is more effective than Citratbuffer.

5.rinsing in PBS

6. Peroxidaseblock. 5-10 min in 3% H2O2 (in water or methanol)

7. rinsing in PBS

8. draw a ring with Dako-Pen (or similar) with sufficient distance to the tissue.

9. Primary Antibody. from Dako - (they sell monoclonal mouse and monoclonal rabbit antibody. which one do you use?) 30 min, RT; in wet chamber, tissue should be covered with sufficient reagens

(Ready to use antibodies usually are diluted in buffer, that needs no additional protein-block)

10. 2x5 min in PBS with agitation

11.20 min secondary-antibody-polymer with Peroxidase. (http://www.dako.com/at/download.pdf?objectid=104958005)

This is a mixture of polymer-conjugated with anti-mouse and anti-rabbit goat antibodies, and can be used with both primary-species.

12. 2x5 min in PBS with agitation

13. 3 min in DAB-solution (described in the link above)

14. rinse in water

15. counterstain in diluted hematoxylin 1-2 min

16. bluing in tap-water

17. dehydration via ethanols 96-100-100; clearing in xylen (or similar), coverslipping

hope this helps

thank you MS Gundrun for your help.....

thank you Mr Dhia

which stage of estrous cycle,you sacrificed  female rats