**2.6 [Ca2+]i fluorescence imaging**

Ca2+ fluorescence imaging was carried out as previously described (Gschossmann *et al.* 2000, Chaban *et al*. 2001). DRG neurons were loaded with fluorescent dye Fura-2 AM (Invitrogen) for 45 min at 37°C in HBSS supplemented with 20 mM HEPES, pH 7.4. The coverslips will be

washed in TBST plus NFDM, and incubated with secondary antibody, HRP conjugated and rabbit IgG (Santa Cruz Biotechnology) at 1:5,000 in the same buffer for 2 hours at room temperature. Following a final wash in TBST without NFDM, the membrane was incubated with ECL+ (Amersham, Arlington Heights, Ill., USA) substrate for HRP. Membranes were probed with primary antibody and corresponding secondary antibodies, signals were scanned and quantified by Image J version 1.28U and NIH Image 1.60 scan software. Following enhanced chemiluminescence (ECL) detection of proteins, the membranes were stripped and rehybridized with β-actin antibody as a loading control. At least three

DRG tissues were obtained from C57/black 6J (The Jackson Laboratory; 30 g), ERαKO and ERβKO (Taconic; 20 g) transgenic types. Following decapitation, DRG from bilateral spinal levels L1-S2 were removed and fixed in 4% paraformaldehyde for overnight at 4oC, according to procedures approved by National Institutes of Health policy on Humane Care and Use of Laboratory Animals. DRGs were rinsed in Delbecco's Phosphate Buffered Saline (DPBS) before cryoprotection in sucrose (20%, 4oC) for two days, after which excess liquid was removed. DRG were quick snap frozen in 2-methylbutane, and store them at -70oC. Each DRG was mounted in Tissue-Tek® OCT embedding medium (Sakura Finetek), and sectioned at - 20oC in a MICROM H505E cryostat. Sections were cut at 20µm and store 4oC until required. Sections of DRGs were collected in PBS. Endogenous tissue peroxidase activity was quenched by soaking the sections for 10 min in 3% hydrogen peroxide solution in 0.01 M PBS. The specimens were washed and then treated for 60 min in blocking solution, 0.01 M PBS containing 0.5% Triton X-100 and 1% normal donkey serum (NDS) at room temperature. They were processed for wild type (n=4), ERαKO (n=4), or ERβKO (n=4) immunohistochemistry by the free floating method using polyclonal rabbit TRPV1 antibody (1:50000, Neuromics) or P2X3 receptor antibody (1:15000, Neuromics) for overnight at 4oC, washed in 0.01 M phosphate-buffered saline (PBS) and 0.01M Tris Buffered Saline (TBS), followed by incubation in solutions of donkey anti-rabbit fluorophore-conjugated secondary antibodies (1:200, Invitrogen) in 0.01M Tris Buffered Saline (TBS) for 3 hours at room temperature. Cells showing no apparent or only faint membrane intracellular labeling were considered to be negative for TRPV1 or P2X3. TRPV1-positive cells included those with strong plasma membrane labeling that formed a discernible clustered pattern, and those with strong intracellular labeling that formed a punctuate pattern. Some neurons showed both strong plasma membrane and intracellular labeling. P2X3-positive neurons showed diffuse membrane intracellular labeling. Mounted sections were air dried and coverslipped with Aqua Poly Mount (Polisciences, Warrington, PA). Images from at least three sections in each level were taken using Leica DMLB M130X microscope. The total numbers of DRG neurons expressing TRPV1 and P2X3 were counted. TRPV1- or P2X3-positive neurons were categorized according to their labeling patterns and were expressed as a percentage of the total number of TRPV1- or P2X3-positive cells. Immunohistochemical signal percent was measured by computerized image analysis

independent cell preparations were used.

(Image Pro-Plus, Media Cybernetics, Silver Spring, MD, USA).

Ca2+ fluorescence imaging was carried out as previously described (Gschossmann *et al.* 2000, Chaban *et al*. 2001). DRG neurons were loaded with fluorescent dye Fura-2 AM (Invitrogen) for 45 min at 37°C in HBSS supplemented with 20 mM HEPES, pH 7.4. The coverslips will be

**2.6 [Ca2+]i fluorescence imaging** 

**2.5 Immunohistochemistry (IHC)** 

mounted on a fast-perfusion chamber P-4 (World Precision Instrument) and placed on a stage of Olympus IX51 inverted microscope. Observations were made at room temperature (20- 23°C) with 20X UApo/340 objective. A fast superfusion system will be used to perfuse the cells with HBSS and rapidly apply E2 and other chemicals. Fluorescence intensity at 505 nm with excitation at 334 nm and 380 nm was captured as digital images (sampling rates of 0.1-2 s). Regions of interest were identified within the soma or neuritis from which quantitative measurements will be made by re-analysis of stored image sequences using Slidebook® Digital Microscopy software. [Ca2+]*i* was determined by ratiometric method of Fura-2 fluorescence from calibration of series of buffered Ca2+ standards. We applied E2 acutely for five minutes onto the experimental chamber or the culture medium for 48 hours to study the prolonged effect of E2. Repeated applications of drugs were achieved by superfusion in a rapid mixing chamber into individual neurons for specific intervals (100-500 ms). Cells were perfused with experimental media (2 ml/min) using a Rainin® peristaltic pump.
