Ab6658

Immunohistochemistry was performed essentially as described previously [38 (link)]. To examine the co-staining of green fluorescent protein (GFP) and Hif1α, the embryos were first stained with goat anti-GFP (Abcam, ab6658, 1:400), and rabbit anti-Hif1α antibody (Novus Biologicals, NB100-134, 1:250) and were subsequently visualized by AlexaFluor-488 donkey anti-goat (Invitrogen, A32814, 1:200) for GFP and AlexaFluor-555 donkey anti-rabbit (Invitrogen, A31572, 1:200) for Hif1α.

Progenies of the intercrossed sart3^+/smu471;Tg(cd41:eGFP)^+/+ transgenic zebrafish were fixed and antibody staining was performed as previously described [72 (link)]. Primary antibody anti-GFP (1:400, ab6658, Abcam, Cambridge, UK) and secondary antibody Alexa Fluor 488-conjugated anti-goat (1:400, A32814, Invitrogen) were used. Images were captured using a Zeiss LSM800 laser scanning confocal microscope and cd41:eGFP^low labeled HSPCs were distinguished from cd41:eGFP^high labeled thrombocytes [46 (link)] by the Range Indicator tool of ZEN software according to fluorescence intensities.

Use 6% SDS-PAGE gel, and the separated proteins were transferred by electro blotting to NC membranes. The membranes were blocked with 5% non-fat dry milk in TBST and incubated with the primary antibody including anti-GFP antibody (Abcam, ab6658) and anti-mCherry antibody (Abcam, ab125096) overnight at 4 °C. Then washing three times, the second antibody including anti-Goat HRP (Proteintech, SA00001-3) and anti-mouse HRP (Proteintech, SA00001-8) for about 2 h Immunolabelling was detected using SuperSignal West Femto (Thermo Fisher Scientific, 34096).

Primary antibodies used were as follows: rabbit anti-GFAP (1:200; Sigma-Aldrich, catalog #G9269; RRID: AB_477035) and biotinylated goat anti-GFP (1:400; Abcam, catalog #ab6658; RRID: AB_305631). Secondary antibodies used were as follows: streptavidin Alexa 488 (1:800; Invitrogen, catalog #S32354; RRID: AB_2315383) for GFP. Goat anti-rabbit antibody conjugated to Alexa 568 (1:400; Thermo Fisher Scientific, catalog #A11011; RRID: AB_143157) for GFAP. Tissue processing for immunohistochemistry was performed as described by Subramanian et al. (2011) (link). Immunohistochemistry for calculating percentage astrocytes in electroporated brains was performed in three biological replicates.

For whole-mount antibody staining, larvae were removed the skin with the Tweezers, washed several times with PT (PBS + 1% Triton X-100) and incubated with primary antibodies below: Anxa4 (1:1000; ab71286, Abcam, Cambridge, MA), 5-methyl-cytosine (1:100; ab10805, Abcam, Cambridge, MA), Alcam (1:50; zn5, ZIRC, Eugene, OR), Bhmt (1:500, a kind gift from Jinrong Peng, Zhejiang University, China), Dendra2 (1:1000; AB821, Evrogen, Moscow, Russia), pS6 (1:500; 2215, Cell Signaling, MA, USA), p4EBP1 (1:500; 2855, Cell Signaling, MA, USA), Dnmt1 (1:200, sc-20701, Santa Cruz Biotechnology, Santa Cruz, CA, a kind gift from Jingwei Xiong, Peking University, China), GFP (1:1000, ab6658, Abcam, Cambridge, MA), DsRed (1:500, sc-101526, Santa Cruz Biotechnology, Santa Cruz, CA) and Tomato (1:1000; orb182397, Biorbyt, TX, USA), After primary antibody incubation, larvae were washed several times with PT and incubated with secondary antibodies conjugated to Alexa Fluor 488/568/633 (1:1000; Invitrogen, Grand Island, NY). The primary and secondary antibodies were diluted in the blocking solution (PBS + 4% BSA + 1% Triton X-100) and incubated at 4 °C overnight and washed with PT for five times.

Fixed brains perfused in dams on day E17.5, or in adult males after GILZ knockdown (KD) injection and phenotyping were serially sectioned and confirmation of the accuracy of the injection site was done by immunostaining using biotinylated α-GFP antibody raised in goat as primary antibody (Abcam ab6658, Cambridge, UK) and streptavidin conjugated Cy2 anti rabbit as secondary antibody (Jackson Immunoresearch laboratories Inc, West Grove, PA, USA).

Two color RNA and protein staining was performed as previously reported (39 (link)). WISH staining was first developed with a TSA plus cyanine 3 kit (Akoya, NEL744001KT). Anti-GFP staining was performed with goat polyclonal anti-GFP primary antibodies (Abcam, ab6658, 1:500) and AF488 donkey anti-goat (Invitrogen, A11055, 1:500) secondary antibodies.