Axioskop fluorescent microscope

Frozen muscle sections (10 μm) were thawed and fixed in 4% paraformaldehyde/PBS for 10 min and blocked in 10% FBS/1% bovine serum albumin (BSA) in PBS (blocking solution) overnight at 4°C. Sections were washed 3 times with PBS and incubated for 2 h in primary antibody (Table 1) diluted in blocking solution, before being washed with PBS and incubated in secondary antibody (Table 2) diluted in blocking solution. Sections were then washed and mounted in Vectashield. Images were captured with a Zeiss Axioskop fluorescent microscope using Spot Advanced software. Images were acquired at the same gain and exposure settings and were processed using the same contrast and brightness settings. The blocking solution for anti-decorin antibody was 10% normal donkey serum in PBS. The isotype control antibodies were rat IgG2a, mouse IgG, rabbit IgG (all from, Life Technologies), and sheep IgG (Jackson ImmunoResearch).

Day 13.5 mouse embryos from WT and Arhgef3 KO were harvested and whole embryo fixation and beta-galactosidase staining was carried out according to the manual protocol from the Cellular Senescence Assay kit (Millipore, Billerica, MA, USA). After beta-galactosidase staining, whole embryos were mounted in tissue TeK (Sakura Finetek, USA) and stored at -80°C. 7μm frozen sections including liver tissue, were mounted onto lysine coated slides (Sigma, St. Louis, MO, USA) and allowed to air dry. Immunohistochemistry was carried out using a primary rat-anti mouse CD41 (BD, USA) and a secondary biotinylated goat anti-rat IgG antibody, and developed using the 3,3’diaminobenzidine (DAB) substrate kit for peroxidase (Vector laboratories, Burlingame, CA, USA). Nuclei were counterstained with nuclear fast red (Sigma, St. Louis, MO, USA) and Digital images captured using an Axiocam (Zeiss, Thornwood, NY, USA) camera mounted onto an Axioskop fluorescent microscope (Zeiss).

The stock colony of Cx. pipiens was reared at 25 °C and 75% relative humidity under a 15-h light:9-h dark (L:D) photoperiod as previously described28 (link). When larvae reached the second instar, rearing containers were placed under one of two environmental conditions: nondiapausing females were generated by rearing at 18 °C, 75% relative humidity, and 15:9 L:D. To induce diapause, mosquitoes were reared at 18 °C, 75% relative humidity, and 9:15 L:D. To confirm diapause status, primary follicle and germarium lengths were measured, and the stage of ovarian development was determined according to the methods described by Christophers29 .
Diapausing and nondiapausing adult females and their fat bodies were collected from 7-days post adult eclosion. Fat body cells were examined by staining fixed tissues with BODIPY 493/503 (Invitrogen). Stained fat body was examined with Zeiss Axioskop fluorescent microscope.

After treatment was stopped, cells were prepared for immunocytochemistry by washing with phosphate buffered saline (PBS) for 5 min, followed by a 20-min fixation with PBS containing 4% (w/v) paraformaldehyde and 4% (w/v) sucrose, pH 7.4 at room temperature. Cells were then permeabilized in PBS containing 0.1% (v/v) Triton X-100 for 5 min, and blocked for 1 h with PBS containing 10% (v/v) goat serum followed by overnight incubation at 4°C with rabbit anti-NFκB (1:1000; α-p65; Abcam), rabbit Iba-1 (1:750; Wako), rat CD11b (1:1000; EBT), rabbit MMP-13 (Abcam; 1:100), or rat CD68 (1:400; Bio-Rad Laboratories) in blocking buffer containing 10% goat serum. Antibody:antigen complexes were visualized following incubation with Alexa Fluor 594 or 488 conjugated goat IgG secondary antibody (1:1000) in PBS containing 0.1% (v/v) triton X-100 and 1% goat serum and subsequently counterstained with 4',6-diamidino-2-phenylindole (DAPI; 13.0 ng/μL) in PBS, followed by two 5 min PBS washes. Coverslips were mounted with Hydromount and cells were imaged and captured using a Zeiss Axioskop fluorescent microscope and AxioCam HRm camera (Carl Zeiss) (Béraud et al., 2011 (link), 2013 (link); Béraud and Maguire-Zeiss, 2012 (link); Daniele et al., 2014 (link), 2015 (link)). Images and subsequent analyses were completed by an observer blinded to treatment.

Primary antibodies were used at the following working concentrations: LC3 (Abgent; 1∶1000), BNIP3 (Sigma; 1∶100). HRP–conjugated anti-rabbit Super Picture (Invitrogen; used for LC3), and anti-mouse ImmPRESS (Vector Laboratories; used for BNIP3) were both used at 1∶100 dilutions. Immunoreactivity was detected using a tyramide signal amplification system using Cy3 (Perkin-Elmer Life Science Products). Bisbenzimide (1 µg/mL; Hoechst 33258; Sigma) was used for nuclear counterstaining. Samples were examined using a Zeiss Axioskop fluorescent microscope equipped with an AxioCam digital camera. Images were captured and analyzed using Axio Vision Rel. 4.8 software (Carl Zeiss MicroImaging).