Two-color Flourescent in situ hybridization (FISH) was carried out as described [29 (link)] with small modifications. Chromosome preparations were denaturated in 70% formamide/2× SSC solution for 5 min at 75 °C and dehydrated in standard series of precooled ethanol at −20 °C. The DNA probes were dissolved in hybridization mixture and denaturated separately for 5 min at 95 °C. Then DNA probes were incubated for 60 min at 37 °C for repetitive DNA renaturation. Hybridization of DNA probes on meiotic chromosomes was carried out overnight in the moist chamber at 37 °C. After hybridization, slides were washed at low stringency condition (3 times for 5 min in 50% Formamide/2× SSC at 45 °C; 3 times for 5 min in 2× SSC at 45 °C and 3 times for 5 min in 0.2× SSC at 45 °C). DAPI counterstaining was preformed after FISH using Vectashield Antifade Mounting Medium containing 4′,6-diamidino-2-phenylindole (DAPI) (Vector labs, Burlingame, CA, USA) applied directly under a coverslip which was then sealed with rubber cement.
Vectashield antifade mounting medium containing 4 6 diamidino 2 phenylindole dapi
Manufactured by Vector Laboratories
Sourced in United States, Canada, Germany
Vectashield antifade mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) is a laboratory product designed to protect fluorescent signals and stain cell nuclei. It is used to prepare samples for microscopy analysis.
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Lab products found in correlation
Normal goat serum (ngs), by Boster Bio (1 mentions)
Alexa fluor 594 conjugated anti rabbit, by Thermo Fisher Scientific (1 mentions)
Tcs sp8, by Leica camera (1 mentions)
Blocking one histo, by Nacalai Tesque (1 mentions)
Mitotracker probe, by Thermo Fisher Scientific (1 mentions)
Lsm 710 confocal laser scanning microscope, by Zeiss (1 mentions)
Axiovert s135 microscope, by Zeiss (1 mentions)
4 protocols using vectashield antifade mounting medium containing 4 6 diamidino 2 phenylindole dapi
1
Two-color Fluorescent In Situ Hybridization
2
Multiplexed Immunofluorescence Staining of Mouse Tumor Tissue
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Paraffin mouse tumor tissue sections were pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 30 min, and blocked with normal goat serum (Cat# AR0009, Boster, China) for 1 h at room temperature. Then, the slides were performed with rat anti-mouse IDO1 (1:100, Cat# 122402, BioLegend), and rabbit anti-mouse CD8 antibody (1:200, Cat# ab203035, Abcam) overnight at 4 °C. After the slides were washed with PBS, they were incubated with Alexa Fluor 633-conjugated anti-rat (1:200, Cat# A-21094, Invitrogen) or Alexa Fluor 594-conjugated anti-rabbit (1:200, Cat# R37117, Life Technologies) secondary antibodies for 2 h in the dark at room temperature. These antibodies were used for labeling the rat anti-IDO1 antibody or the rabbit anti-CD8 antibody, respectively. Slides were mounted using VECTASHIELD antifade mounting medium containing 4,6-diamidino-2-phenylindole (DAPI) (Cat# H-1200, Vector Laboratories) according to manufacturer’s recommendations. Fluorescent staining was visualized under a laser scanning confocal microscope (TCS SP8, Leica, Germany).
3
Adipocyte Mitochondrial Dynamics Visualization
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3T3-L1 adipocytes plated on cover glasses were cultured in DMEM containing GluOC for 12 or 24 h and, where indicated, then loaded with the MitoTracker probe (Invitrogen, Carlsbad, CA) for 45 min at 37 °C. The cells were then fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.2% Triton X-100 for 5 min at room temperature. After exposure to Blocking One histo (Nacalai Tesque) for 5 min at room temperature, the cells were incubated with antibodies to FasL (Abcam) or with those to N-cadherin or to DRP1 (Cell Signaling Technology) for 1 h and then with Alexa Fluor 488- or Alexa Flour 546-conjugated secondary antibodies (Thermo Fisher Scientific) for 30 min at room temperature. The cover glasses were then inverted onto glass slides, to which Vectashield antifade mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA) was applied for 30 min at room temperature. The cells were then observed with an LSM710 laser-scanning confocal microscope (Carl Zeiss, Oberkochen, Germany). All confocal images are representative of at least three independent experiments.
4
Fetal Origin Verification of CV-MSCs
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To confirm the fetal origin of the CV-MSC isolated from placentas collected after the birth of male newborns, X/Y chromosome analysis was performed at early (p3–p4) and late (p8–p10) passages for all donors used for different subsequent experiments (n = 5) (Fig. 1a iv–v). The ZytoLight CEN X/Y Dual Color Probe (Zytomed Systems, Germany) was used for detection of human α-satellites of X and Y chromosomes by fluorescence in-situ hybridization (FISH). Ten microliters of the hybridization mixture was added to the cytospun cells, and DNA denaturation was then performed at 75 °C for 2 minutes with subsequent overnight incubation using a humidified chamber. After hybridization, the cytospins were washed with cytology stringency buffer for 2 minutes at 72 °C and subsequently rinsed in sodium chloride/sodium citrate buffer for 1 minute at room temperature (RT). Slides were finalized using Vectashield antifade mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) (Vector Labs, Germany) and fluorescence detected on an Axiovert S135 microscope (Zeiss, Germany). The α-satellite sequences of the centromere of chromosome X were excited at 488 nm and of chromosome Y at 554 nm.
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