Alexa fluor 647 donkey anti rabbit

Adult hearts with pericardial nephrocytes from 7-day-old females were dissected in artificial Drosophila haemolymph according to Selma-Soriano et al. (2018a) (link), fixed with 4% paraformaldehyde (PFA) in PBS for 20 min and permeabilized by washing three times with PBS containing 0.3% Triton X-100 (PBS-T) for 10 min. Then, hearts were blocked in PBS-T containing 0.5% bovine serum albumin for 30 min at room temperature and incubated with the corresponding primary antibody (1:100) overnight at 4°C. Primary antibodies used were anti-Rph (Abcam, ab3338; with 50% of homology), anti-Hrs (DSHB, AB 2722114), anti-Rab3 and anti-Rab27 (BD Bioscience, 610379 and 558532, respectively) and anti-Klf15 (Ivy et al., 2015 (link)) (Abcam, ab22851; with 59% of homology). After three washes with PBS-T, the secondary antibodies (1:200), AlexaFluor-647 donkey anti-rabbit (Life Technologies, A31573), anti-mouse biotinylated (Sigma-Aldrich, B7264) and anti-rabbit biotinylated (Sigma-Aldrich, B8895) were incubated for 2 h at room temperature. Hearts with pericardial nephrocytes were then incubated with ABC solution (ABC kit, VECTASTAIN) for 30 min at room temperature, followed by washes and incubation with streptavidin-Texas Red (Vector Laboratories, 1:500). All images were taken using an LSM 800 confocal microscope (Zeiss) and were processed using ZEN software.

The following antibodies were used: GFP (ab13970; Abcam), GFP (ChIP; ab290) CycA2 (#sc-751; Santa Cruz) (used for immunofluorescence), CycA2 (#4656; Cell Signalling), CycA2 (HPA020626), PLK1 (ab14210; Abcam), affinity purified mouse anti pT210-PLK1 (clone K50-483; Becton Dickinson), affinity purified rabbit anti-Bora (Bruinsma et al, 2014 (link)), CDK1 (sc-54; Santa Cruz and #9116; Cell Signalling), CDK2 (sc-163; Santa Cruz and #2564; Cell Signalling), GAPDH (G9545; Sigma-Aldrich), H2B (ab1790; Abcam), β-Tubulin (#2128S; Cell Signalling), PCNA (HPA030522), KAP1 (A300-274A), H3 (ab1791), Alexa Fluor 488–goat anti-chicken (#A11039; Life Technologies), and Alexa Fluor 647–donkey anti-rabbit (#A31537; Life Technologies).

Adult hearts from 7-day-old females were dissected in PBS 1 X according to Selma-Soriano and Chakraborty³⁸ , fixed with 4% paraformaldehyde for 20 min and permeabilized by PBS containing 0.3% Triton-X (PBS-T) for 10 min, 3 times. Hearts were blocked in PBS-T containing 0.5% BSA for 30 min at room temperature and incubated with human anti-Rabphilin (1:200) (Abcam, ab3338). After 3 washes with PBS-T, the AlexaFluor-647 donkey anti-rabbit (1:1000) (Life Technologies, A31573) was incubated for 2 h at room temperature. The images were taken with an LSM 800 confocal microscope (Zeiss) using 40 × oil objective.

N2A cells were co-transfected with PIEZO2-HA-IRES-GFP or IDRdel constructs and with a plasmid encoding the red fluorescent protein mScarlet fused to a farnesylation signal sequence in its C-terminus to target it to the plasma membrane. Three days after transfection, cells were washed once with PBS and fixed with 4% PFA for 10 min at room temperature, washed 3 times for 5 min with PBS and permeabilised for 1 h at room temperature (permeabilization buffer: 2,5% donkey serum (Sigma), 1% BSA, 0.1% Triton X-100, 0.05% Tween-20, in PBS). Samples were then incubated overnight at 4 °C with a 1:500 dilution of rabbit anti-HA antibody (Thermo Fisher Scientific) in PBS 1% BSA. After 3 washes of 5 min, cells were incubated for 1 h with a 1:1000 dilution of AlexaFluor-647 donkey anti-rabbit (Life technologies, diluted in PBS 1% BSA) and washed 3 more times. Coverslips were mounted on slides with FluoProbe mounting media that contain DAPI (Interchim). Confocal images were acquired with a SP8 confocal microscope (Leica) and a 63x oil-immersion objective. Images were analyzed offline with Fiji (v2.3.0/1.53f).

Paraffin sections of white adipose tissue (5μm thickness) were stained with rat anti mouse Ly6G (1:100; BD Biosciences) and rabbit anti mouse Cathelicidin (1:100; Innovagen). Subsequently, samples were incubated with Alexa fluor 647 donkey anti rabbit (1:500; Invitrogen), donkey anti rat 549 (1:500; Invitrogen), and the DNA-binding dye DAPI (Invitrogen). Co-localization of these markers was determined by confocal microscopy (SP-8 X, Leica). In addition paraffin sections of white adipose tissue were stained with hematoxylin and eosin. Adipocyte size was determined of 100 adipocytes per mouse by use of ImageJ.