Goat anti agt

Paraffin sections of iERM tissues were deparaffinized and hydrated through exposure with xylene and graded alcohols followed by water. As a pretreatment, microwave-based antigen retrieval was performed in 10 mM citrate buffer (pH 6). Sections were incubated with the following primary antibodies: rabbit anti-(P)RR (Sigma-Aldrich), mouse anti-prorenin, rabbit anti-ACE and rabbit anti-AT2R (Abcam, Cambrige, MA, USA), rabbit anti-AT1R and goat anti-AGT (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-FGF2 (Millipore, Temecula, CA, USA), goat anti-GDNF, goat anti-NGF, mouse anti-TGF-β1 and mouse anti-α-SMA (R&D systems, Minneapolis, MN, USA), and mouse anti-GFAP (Leica, Exton, PA, USA) antibodies. Secondary antibodies for fluorescent detection were AlexaFluor 488 and 546 (Life Technologies). Sections were examined using the Keyence BZ-9000 (Keyence, Osaka, Japan).

TM tissue samples surgically excised from patients were embedded in paraffin after fixation in 4% paraformaldehyde. Paraffin sections of TM tissues were dewaxed, rehydrated, and rinsed in PBS. As a pretreatment, microwave-based antigen retrieval was carried out in 10 mM citrate buffer (pH 6.0). Sections were probed with the following primary antibodies: rabbit anti-(P)RR (Sigma-Aldrich), mouse anti-prorenin (Abcam), rabbit anti-AT1R, goat anti-AGT, goat anti-tissue plasminogen activator (t-PA), goat anti-placental growth factor (PlGF) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-connexin (CX)43, and rat anti-zona occludens (ZO)-1 (Millipore, Temecula, CA, USA) antibodies. Normal mouse, goat, and rabbit IgGs were used as negative control antibodies. Secondary antibodies for fluorescent detection were labeled with Alexa Fluor 488 and Alexa Fluor 546 (Thermo Fisher Scientific, Waltham, MA, USA). Sections were visualized under the BIOREVO BZ-9000 fluorescence microscope (Keyence, Osaka, Japan).