The following antibodies were applied: Tetraspanin-4 (NBP1-59438, Novus); RPE 65 (MA1-16578; Thermo Fisher Scientific); GFAP (ab279290, Abcam); CD11b (ab8878, Abcam); integrin α5 (ab288767, Abcam); EOGT (ab190693, Abcam); NDST1 (26203-1-AP, Proteintech); Alix (2171, Cell Signaling); Alix (92,880, Cell Signaling); Smad2/3 (3102, Cell Signaling); Phospho-Smad2/Smad3 (8828, Cell Signaling); Alpha-Smooth Muscle Actin (MA5-11547, Thermo Fisher Scientific); GAPDH (5174, Cell Signaling) and β-actin Rabbit antibodies (ab8227, Abcam). Secondary antibodies used included the following: Alexa Fluor 488 Anti-Mouse; Alexa Fluor 555 Anti-Rabbit; Alexa Fluor 555 Anti-Mouse; and Alexa Fluor 555 Anti-Rabbit (Thermo Fisher Scientific). Additionally, CCK8 (ab228551, Abcam), Dulbecco’s modified Eagle’s medium (DMEM)/F12 culture media, and fetal bovine serum were obtained from Thermo Fisher Scientific.
Ab228551
Manufactured by Abcam
Ab228551 is a laboratory equipment product. It is a tool used for scientific research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Ab8878, by Abcam (1 mentions)
Ab279290, by Abcam (1 mentions)
Smad2 3, by Cell Signaling Technology (1 mentions)
Phospho smad2 smad3, by Cell Signaling Technology (1 mentions)
Anti mouse alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Cck 8, by Abcam (1 mentions)
Rpe 65, by Thermo Fisher Scientific (1 mentions)
β actin rabbit, by Abcam (1 mentions)
Alexa fluor 555 anti rabbit, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Ab8227, by Abcam (1 mentions)
Ab32527, by Abcam (1 mentions)
Ab6939, by Abcam (1 mentions)
Ab16502, by Affinity Biosciences (1 mentions)
Tx 100, by Beyotime (1 mentions)
Ab9465, by Abcam (1 mentions)
Ab33589, by Abcam (1 mentions)
Ab150118, by Abcam (1 mentions)
7 protocols using ab228551
1
Immunostaining of Cellular Markers
2
Immunostaining of TSCs and Macrophages
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TSCs, cocultured with macrophage, were digested and collected for cell slit. Palovarotene or LDN193189 and induction treatment were given according to the experimental group for 30 min. After washing the cells with PBS, 4% paraformaldehyde (Biosharp, # BL539A) was added, and the cells were fixed after 20 min at room temperature. PBS washes were given twice again, and 0.1% Tx-100 (Beyotime, # P0096) was added. After 15 min at room temperature, the liquid turned transparent, and 5% BSA/PBS was added, and the cells were incubated at 37°C for 1 h. The TPPP3 (Biorbyt, #5-F10) or CCR7 (Abcam, # ab32527) or p65 (Abcam, # ab16502) or Smad5 (Affinity, #AF5119) or Id1 (Proteintech, #18475-1--AP) antibody working fluid was added, and the cells were incubated at 4°C overnight. After overnight incubation, the samples were washed 3 times in PBS and were incubated at 37°C for 1 h with anti-rab-CY3 (ABCAM, #AB6939) working solution. The samples were washed 3 times in PBS and were incubated at room temperature for 15 min with Hoechst (ABCAM, #AB228551) and were washed 3 times in PBS. Lastly, the samples were seeded and photographed under a fluorescence microscope.
3
Cardiac Muscle Cell Immunostaining
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At
14 days of culture, samples were rinsed with PBS, and cells were fixed
with 4 wt % paraformaldehyde (PFA) at room temperature for 30 min.
After fixation, cells were permeabilized with 0.1% Triton X-100 for
5 min and blocked with 1 wt % bovine serum albumin containing 22.52
mg/mL glycine prepared in PBS for 30 min. Then, samples were incubated
in anti-sarcomeric α-actinin (1:50 dilution, ab 9465, Abcam)
and monoclonal anti-cardiac troponin T (cTnT) (1:25 dilution, ab33589,
Abcam) primary antibodies for 4 h at room temperature.41 (link) Afterward, samples were rinsed with PBS and
incubated in Alexa Fluor 555 (1:1000, ab150118, Abcam) and Alexa Fluor
488 (1:1000, ab150113, Abcam) secondary antibodies for 1 h to stain
α-actinin and cTnT, respectively. The actin cytoskeletons of
the cells were stained with Alexa Fluor 488 Phalloidin (Thermo Fisher).
The nuclei of cells were counterstained with 25 mM Hoechst (Abcam,
ab228551). Images were acquired using a confocal microscope (DMI 8,
Leica), and processed using Leica LASX software.
14 days of culture, samples were rinsed with PBS, and cells were fixed
with 4 wt % paraformaldehyde (PFA) at room temperature for 30 min.
After fixation, cells were permeabilized with 0.1% Triton X-100 for
5 min and blocked with 1 wt % bovine serum albumin containing 22.52
mg/mL glycine prepared in PBS for 30 min. Then, samples were incubated
in anti-sarcomeric α-actinin (1:50 dilution, ab 9465, Abcam)
and monoclonal anti-cardiac troponin T (cTnT) (1:25 dilution, ab33589,
Abcam) primary antibodies for 4 h at room temperature.41 (link) Afterward, samples were rinsed with PBS and
incubated in Alexa Fluor 555 (1:1000, ab150118, Abcam) and Alexa Fluor
488 (1:1000, ab150113, Abcam) secondary antibodies for 1 h to stain
α-actinin and cTnT, respectively. The actin cytoskeletons of
the cells were stained with Alexa Fluor 488 Phalloidin (Thermo Fisher).
The nuclei of cells were counterstained with 25 mM Hoechst (Abcam,
ab228551). Images were acquired using a confocal microscope (DMI 8,
Leica), and processed using Leica LASX software.
4
Immunofluorescence Staining of Cas9 and HBc
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Cells seeded on glass coverslips were fixed in 4% paraformaldehyde for 10 min. Next, coverslips were washed three times in 50 mM Tris-HCl (pH 8.0), incubated for 30 min with blocking buffer (0.02% Triton X-100, 10% horse serum, and 150 mM NaCl in 50 mM Tris-HCl [pH 8.0]), and incubated with primary goat polyclonal anti-6XHis-tag antibodies to detect StСas9-6XHis-tag protein (ab9136, Abcam) and with mouse monoclonal anti-HBc antibodies (ab8637, Abcam) at room temperature for 1 h. The cells were washed three times for 5 min in washing buffer (0.02% Triton X-100 and 200 mM NaCl in 50 mM Tris-HCl [pH 8.0]), then incubated with secondary Alexa Fluor 647 donkey anti-goat immunoglobulin G (IgG) H&L (ab150131, Abcam) and Alexa Fluor 488 donkey anti-mouse IgG H&L antibodies (ab150105, Abcam). Alternatively, coverslips were stained only with primary mouse monoclonal anti-HBc antibodies (ab8637, Abcam) and secondary Alexa Fluor 594 goat anti-mouse IgG H&L antibodies (ab150116, Abcam). Cell nuclei were counterstained with Hoechst 33342 (1/10,000; ab228551, Abcam) at room temperature for 1 h. Coverslips were washed three times for 5 min in washing buffer and mounted with Fluoroshield reagent (Abcam). Images were captured using a Leica DMI6000 microscope with 100× immersion objectives. The research was done using equipment of the Core Centrum of Institute of Developmental Biology RAS.
5
Whole-Mount Adipose Tissue Imaging
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For whole-mount fluorescence microscopy of adipose tissue, fascia was carefully removed from inguinal fat pads under a dissecting microscope and the tissue was then fixed for 1 h at room temperature with 1% paraformaldehyde in PBS and permeabilized for 1 h with 0.3% Triton X-100 in PBS (PBST) before incubation for 30 min at room temperature with Hoechst 33342 (H3570, Invitrogen) at a dilution of 1:1,000 and boron dipyrromethene (BODIPY; D3822, Invitrogen) at a dilution of 1:2,000 in PBST. The tissue was washed several times with PBST, mounted in fluorescence mounting medium (S3023, Dako) and imaged with a confocal microscope (LSM880, Carl Zeiss). For Oil Red O staining of lipid droplets, cells were fixed with 4% formaldehyde, stained with Oil Red O solution (O1516, Sigma) for 20 min, and washed with PBS. Fluorescence imaging of cells was performed after staining with Hoechst 33342 (ab228551, Abcam) and BODIPY 493/503 (D3922, Thermo Fisher Scientific). Lipid droplet accumulation was assessed at 6 days after application of the adipogenic cocktail to induce adipocyte differentiation. The cells were imaged using a CQ1 microscope (Yokogawa).
6
Multicolor Imaging of Adipocytes
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Differentiating ASPCs (day 6) from supraclavicular BAT of one donor were fixed in a standard 96-well plate as described in RNA FISH. After performing RNA FISH staining for DCN and ADIPOQ, the cells were permeabilized with 0.1% Triton X-100 (1003407653, Sigma) in PBS for 10 min at room temperature (RT), blocked with 10% goat serum (G9023, Sigma) in PBS for 60 min at RT and incubated overnight at 4 °C with the primary anti-perilipin antibody (9349S, Cell Signaling) in blocking solution (1:200 dilution). After 3× thorough PBS washes, Alexa Fluor 488-conjugated secondary antibody (A21206, Invitrogen) was diluted at a ratio of 1:750 in PBS and added for 1 h at RT followed by 2× PBS washes. Nuclei were stained with Hoechst 33342 (8 µM; ab228551, Abcam) for 10 min at RT and washed 3× with PBS, whereas the last wash was left on the cells. Images were acquired with the Leica Thunder microscope at a magnification of ×20.
7
Immunofluorescence Staining of Beta-Actin
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Cells were cultured as described above. After 72 h, the media was discarded, the cells quickly rinsed in phosphate-buffered saline (PBS) solution (pH 7.4) and fixed in 4% formaldehyde (1.04003.2500 –MilliporeSigma) in PBS (pH 7.4) for 20 min at RT. Then, the cells were permeabilized in 0.01% triton in PBS (pH 7.4) for 15min at RT, blocked in 3% bovine serum albumin (BSA) in Tris-buffered saline (TBS) solution (pH 7.6) for 1h at 4°C and incubated ON at 4° C with a FITC anti-beta actin antibody (ab6277—Abcam) 1:100 in 3% BSA in TBS (pH 7.6). The cells were washed 3 times for 5 min in PBS (pH 7.4) at RT and incubated for 5 min with Hoechst 33342 (ab228551—Abcam) 1:5000 in PBS (pH 7.4) at RT. Finally, the cells were washed 3 times for 5 min in 0.1% Tween in PBS (pH 7.4) at RT and rinsed 1 time in PBS (pH 7.4) at RT.
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