Third instar larvae were dissected in PBS to prepare lymph gland samples as described before (Khadilkar et al., 2014 (link)). Samples were fixed with 4% paraformaldehyde (PF) for 20 min at room temperature (25°C), permeabilized with 0.3% PTX (Triton X-100 in PBS) and incubated in 20% goat serum before primary antibody addition. Antibodies used were mouse anti-COX IV (Abcam, United Kingdom), rabbit anti-Asrij (Kulkarni et al., 2011 (link)), mouse anti-P1 (Istvan Ando, BRC Hungary), mouse anti-ProPO, rabbit anti-dsRed (Takara, Japan), and chick anti-GFP (Abcam, United Kingdom).
For hemocyte immunostaining, larvae were bled to extract hemolymph into warm Schneider’s serum-free media (Thermo Fisher Scientific, Waltham, MA, United States). Hemocytes were placed on coverslips to allow attachment for 10 min, then fixed with 4% PF and permeabilized with 0.4% NP40, blocked with 20% goat serum and incubated in primary antibody.
Secondary antibodies used were conjugated to Alexa-Fluor 488, 568, or 633 (Life Technologies, Carlsbad, CA, United States). Phalloidin conjugated to Alexa 568 or 633 (Life Technologies, Carlsbad, CA, United States) was used to visualize lamellocytes. Lymph glands were mounted on coverslips in DAPI-glycerol media. Images were acquired using Zeiss LSM510 Meta or LSM880 confocal microscope in either normal confocal mode or airy scan mode.
For hemocyte immunostaining, larvae were bled to extract hemolymph into warm Schneider’s serum-free media (Thermo Fisher Scientific, Waltham, MA, United States). Hemocytes were placed on coverslips to allow attachment for 10 min, then fixed with 4% PF and permeabilized with 0.4% NP40, blocked with 20% goat serum and incubated in primary antibody.
Secondary antibodies used were conjugated to Alexa-Fluor 488, 568, or 633 (Life Technologies, Carlsbad, CA, United States). Phalloidin conjugated to Alexa 568 or 633 (Life Technologies, Carlsbad, CA, United States) was used to visualize lamellocytes. Lymph glands were mounted on coverslips in DAPI-glycerol media. Images were acquired using Zeiss LSM510 Meta or LSM880 confocal microscope in either normal confocal mode or airy scan mode.
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