Fluoromount g mounting media

We washed saccule, utricle, and lagena sensory epithelia twice with 0.1M phosphate buffered saline (PBS) and incubated them for 1 hour with phalloidin conjugated to rhodamine (Thermo Fisher Scientific Invitrogen, cat. no. R415) diluted 1:40 in 0.1 M PBS. We washed epithelia again with 0.1 M PBS and then placed them on coverslips (oriented with apical sides against the coverslip) and whole mounted them in Fluoromount-G mounting media (Southern Biotech, cat. no. 0100–01) onto slides. We sealed slides with nail polish and stored them at +4° C in the dark until fluorescence imaging.

After treatments, platelets were spread on fibrinogen-coated coverslips as a surrogate measure of wound healing, as previously described [32 (link)]. Coverslips were coated with 50 μL of 250 μg/mL fibrinogen (Sigma-Aldrich, St Louis, MO) for 2 hours at room temperature, then blocked with 0.5% bovine serum albumin (BSA; Sigma-Aldrich, St Louis, MO) in 1 X phosphate buffered saline (PBS) for up to 1.5 hours. Washed human platelets were diluted to 3.3 × 10⁷ platelets/mL in TSS and 300 μL plated onto the fibrinogen coated coverslips, then incubated at 37°C for 45 minutes to spread. The coverslips were then washed with PBS three times, and fixed with 4% paraformaldehyde in PBS for 15 minutes. After fixing, coverslips were washed once with PBS, then mounted onto slides using Fluoromount G mounting media (Southern Biotech). Images were taken at 100X with an Olympus BX51 microscope (Olympus, Melville, NY) using differential interference contrast microscopy (DIC), and at least 5 fields of view were captured for each coverslip. Images were edited using SPOT software to enhance platelet visibility. Platelet spreading was quantified by manual counting, after blinding via randomizing images and obscuring sample information.

Subgroups of hearts were harvested at 4 and 14 weeks after TAC for analyses of structure, fibrosis, and biochemistry. Trichrome staining was performed as previously described (44 (link)). Briefly, mice were euthanized, and after cardioperfusion, hearts were fixed for 1 to 3 days in 4% paraformaldehyde at 4°C. Hearts were dehydrated and paraffinized using the Microm STP 120 from Thermo Fisher Scientific, embedded in paraffin using a HistoStar apparatus (Thermo Fisher), and sectioned (4 to 6 μm) using the Microm HM 325 (Thermo Fisher). Tissue sections were then stained with Weigert’s iron hematoxylin and Masson trichrome (Sigma-Aldrich) according to the manufacturer’s instructions. Interstitial fibrosis was quantified by color threshold measures using ImageJ. Alternatively, tissue sections were deparaffinized and rehydrated according to the trichrome staining protocol, but after the wash with deionized water, heart sections were washed three times for 5 min with 1× phosphate-buffered saline (PBS) followed by staining with Alexa Fluor 488–conjugated WGA (1:10 in 1× PBS) (Invitrogen) for 1 hour at room temperature in a humidified chamber in the dark. Sections were again washed three times for 5 min with 1× PBS followed by mounting with coverslips using Fluoromount-G mounting media containing DAPI nuclear stain (Southern Biotech).

ST3^{ΔLRR9/ΔLRR9} and WT littermate mice at P21–P25 were anesthetized with isoflurane, perfused transcardially with 4% PFA + 0.1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4 (PB). Brains were then dissected out, post-fixed in the same solution for 1 h at RT, and then cryoprotected overnight in 30% sucrose in 1× PB at 4°C. Coronal brain sections (40 μm) were collected at −20°C with a cryostat (Leica CM1050). After washing with 1xPBS, brain sections were incubated in the blocking solution (0.2 % Triton X-100 and 5 % normal goat serum in 1xPBS) for 1 h at RT under gentle agitation, and followed by incubation at 4°C with primary antibodies in 1xPBS containing 0.2 % Triton-X with 3 % normal goat serum (anti-vGAT, 1:4000, rabbit SYSY; anti-Gephyrin, 1:1000 mouse, SYSY) for 48 h. Brain sections were then washed 3 times (20 min each time) in 1xPBS, incubated with secondary antibodies (1:1000, Alexa 488, 555, Thermo Fisher Scientific) for 1 h at RT, and followed by washing for 3 times (20 min each time) in 1xPBS. Brain sections were then mounted on superfrost slides and covered with DAPI-containing Fluoromount G mounting media (Southern Biotech).