Hi, we use the DAKO antibody M7047 mouse anti human at a concentration of 1:200 on our sheep sections. Works a treat. We antigen retrieve in citrate buffer pH 6 for sheep pituitary sections but not sure if you need to do this for ovary. Good luck! Oh and its for ERalpha!

Thanks Michelle,

Peter see our paper

Okumu LA, Forde N, Fahey AG, Fitzpatrick E., Roche JF, Crowe MA and Lonergan P (2010). The effect of elevated progesterone and pregnancy status on mRNA expression and localisation of progesterone and oestrogen receptors in the bovine uterus. Reproduction 140: 143-153.

This work was in cattle, but should work in cattle. This paper is available in full on my Research Gate page

Our methods were as follows:

Immunohistochemistry of ESR1, ESR2, PGR-AB and PGR-B Protein localisation and intensity were determined using IHC analysis, using a previously described procedure (Van den Broeck et al. 2002), which we optimised to suit the current experiment. Unless otherwise stated, all washes were done twice in 0.05 M Tris-buffered saline (TBS; pH 7.7) for 5 min each, and all incubations were carried out in a humid chamber at room temperature. Four micron-thick sections of whole uterine tissue were cut from the paraffin-embedded tissue blocks, mounted on glass slides coated with 98% 3-aminopropyl triethoxysilane and dried overnight at 56 8C. One block per animal was stained, and there were five animals per treatment per time point as outlined above. From each block (i.e. animal), 15 sections were taken; from amongst these 15 sections, sections were chosen at random for testing the various antibodies. Two negative control slides (where the primary antibody was omitted) and a pre-determined positive control slide were also included in each assay. Slides were deparaffinised by two washes in xylene and rehydrated through a series of graded alcohol steps (100 and 70%). Antigen retrieval was achieved by heating the slides in 0.01 M sodium citrate buffer (May & Baker Ltd, Essex, UK) at a pH of 6.0 for 20 min, and subsequent cooling for a further 20 min.

Endogenous peroxidase activity was neutralised by incubating

the slides for 30 min with 1% hydrogen peroxide in

methanol. Non-specific binding was inhibited by incubating

the slides for 30 min with 2% of either normal rabbit serum (for

both PGR (Laboratory Instruments and Service Centre,

Ashbourne, Co., Meath, Ireland) and ESR2 (Dako Diagnostics,

Cambridgeshire, UK) antibodies) or goat serum (for ESR1

antibody: Laboratory Instruments and Service Centre) in

0.05 M TBS (v/v). Primary antibodies (ESR1, PGR-AB and

PGR-B sourced from Laboratory Instruments and Service

Centre; and ESR2 sourced from Dako Diagnostics) were

incubated at the appropriate dilution and under optimum

conditions (Table 2). Slides were then washed and incubated

for 45 min with the polyclonal biotinylated secondary

antibodies, which was a rabbit anti-mouse at a 1:100 dilution

(for PGR-AB, PGR-B and ESR2) and a goat anti-rabbit at a

1:300 dilution (for ESR1; Dako Diagnostics). The slides were

washed, and avidin–biotin–HRP complex (Vectastain Elite ABC

Kit, Vector Labs, Peterborough, UK) was added to the slides and

they were incubated for 30 min. Following further washing,

3,30-diaminobenzidine tetrahydrochloride chromogen substrate

was added to the slides, and they were incubated for

10 min. The above-mentioned reaction was stopped by

flushing the slides with distilled water, and the slides were

then washed under running tap water for 7 min. This was

followed by dehydration using increasing concentrations of

alcohol (70 and 100%) for 5 min each and subsequent clearing

in two successive changes of xylene for 10 min each. Slides

were mounted using DPX (AGB Scientific Ltd, Dublin, Ireland)

and observed under 10! magnification. Using a digital camera, four images were captured per tissue section (two

images showing the LE, SGs and STR, and two images showing

the DGs and MYO). Intensity of staining for all the regions was

determined using Image-Pro Plus software (version 6.2,

MediaCybernetics, Bethesda, MD, USA).

Hi Mark,

Thanks, that's very helpful and a nice paper in Reproduction. I notice the Dako Ab (clone ppg5/10) to ESR-2 detects across a range of species.. Co-incidentally we are about to try a Dako ESR-1 Ab on wax embedded sheep ovary so if the ESR-2 also works we will be very pleased.

cheers

Peter

My colleagues from INRA Tours published some years ago the IHC detection of ER with 2 monoclonals (Tomanek et al, MOLECULAR REPRODUCTION AND DEVELOPMENT 1997, 48:53–62 ). You may ask directly to Danielle.Monniaux @tours.inra.fr.