Goat anti rat igg h l alexa fluor 568

Fixation of C. reinhardtii was performed using a modified method in a previous study [34 (link)]. C. reinhardtii cells were attached to polyethyleneimine coated coverslips and fixed in −20 °C methanol for 5 min, transferred to fresh −20 °C methanol for 5 min and air-dried. The dried cells were incubated in PBS for 10 min. Subsequent blocking and antibody reactions were performed based on a previous study [5 (link)]. Immunostaining of T. socialis and G. pectorale were performed as described previously [5 (link)]. The anti-TsDRP1 antibody and a monoclonal anti-tubulin alpha antibody (clone YL1/2, Bio-Rad, Hercules, CA, USA), used as primary antibodies, were diluted 1: 500 with blocking buffer (0.11% Gelatin [Sigma Aldrich], 0.05% NaN₃, 0.25% bovine serum albumin [Sigma Aldrich] in TPBS). Alexa Fluor 488 goat anti-rabbit IgG (H + L) (# A11008, Invitrogen, Carlsbad, CA, USA) and Alexa Fluor 568 goat anti-rat IgG (H + L) (# A11077, Invitrogen) were also diluted 1: 500 with the blocking buffer. Confocal and differential interference contrast (DIC) images were obtained with an FV-1200 (Olympus, Tokyo, Japan) and three serial images were merged by using Adobe Photoshop CS6 software (Adobe Systems Inc., San Jose, CA, US).

The ICC were stained for c-kit, as described previously (Horvath et al., 2005 (link)). Fundus was pinned flat and fixed with 100% acetone at room temperature for 30 min. After washing twice with water, the tissue was gently shaken in 1 × PBS overnight at 4°C. Next, the tissue was incubated in PBS with 1% BSA and 0.3% Triton X-100 for 2 h at room temperature with gentle shaking and then washed twice with PBS plus 1% BSA for 20 min. Tissue was incubated for 48–72 h at 4°C with gentle shaking in anti-c-kit ACK2 antibody (1:20 in PBS, 1% BSA). The tissue specimen was washed 6 times for 7 min each in PBS plus 1% BSA at room temperature on the shaker. Alexa fluor 568 goat anti-rat IgG H+L (2 mg/ml Invitrogen, Carlsbad, CA) was used as the secondary antibody (1:250 in PBS, 1% BSA). After incubation for 2 h at room temperature, the tissue specimens were washed 5 times and mounted on slides with Fluorosave for fluorescence imaging by an Olympus BX51 microscope powered by DP Controller imaging system.