For BrdU (5-bromo-2′-deoxyuridine) labelling, mice were intraperitoneally injected with 10 μl per gram body weight of BrdU labeling reagent (#000103, Invitrogen) 6 h before being sacrificed. The hindlimbs were then harvested and processed for paraffin sections. To detect BrdU-labeled proliferating cells, tibial sections were dewaxed, rehydrated, and then denatured with 2 N HCl, prior to antigen retrieval with 0.125% trypsin in PBS at 37°C for 10 min. After blocking with 10% goat serum for 1 h, sections were incubated with rat anti-BrdU antibody (ab6326, Abcam, 1:100) at 4°C overnight, followed by incubation with Alexa Fluor 647-conjugated donkey anti-rat (ab150155, Abcam,1:200) or Alexa Fluor 488-conjugated goat anti-rat secondary antibody (ab150157, Abcam, 1:200). Finally, nuclei were counterstained with DAPI (C1005, Beyotime Biotechnology, Shanghai, China) to stain the cell nuclei, and subsequently mounted with anti-fade mounting medium.
Ab150155
Manufactured by Abcam
Ab150155 is a laboratory reagent product manufactured by Abcam. It is designed for use in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab150135, by Abcam (2 mentions)
Ab150073, by Abcam (2 mentions)
Ab18465, by Abcam (2 mentions)
Brdu labeling reagent, by Thermo Fisher Scientific (1 mentions)
Ab150157, by Abcam (1 mentions)
Ab6326, by Abcam (1 mentions)
Facsmelody, by BD (1 mentions)
Intracellular staining perm wash buffer, by BioLegend (1 mentions)
C6 plus, by BD (1 mentions)
Fixation buffer, by BioLegend (1 mentions)
Ma5 16987, by Thermo Fisher Scientific (1 mentions)
Rat anti epcam, by BD (1 mentions)
Rabbit anti laminin, by Merck Group (1 mentions)
Goat anti mouse perilipin, by Abcam (1 mentions)
Rat anti mouse cd31, by BioLegend (1 mentions)
Cm3050s cryostat, by Leica (1 mentions)
Tcs sp8, by Leica (1 mentions)
Optimal cutting temperature compound, by Sakura Finetek (1 mentions)
A21206, by Thermo Fisher Scientific (1 mentions)
A10036, by Thermo Fisher Scientific (1 mentions)
11 protocols using ab150155
1
Quantifying Cell Proliferation using BrdU Labeling
2
Flow Cytometry Identification of Macrophages and Somatostatin-Expressing Cells
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The cells were suspended in PBS (containing 1% BSA) and then incubated with antibody (CD68, 1:100, 14-0688-82), at 4°C for 30 min. CD68 was used to sort macrophages (19 (link)), which were then washed twice with PBS containing 1% BSA. Then, the cells were incubated with Alexa Fluor 488-conjugated donkey anti-mouse IgG (1:500, ab150117, RRID: AB_2688012) at 4°C in the dark for 30 min and washed twice with PBS containing 1% BSA. The cells were then resuspended in PBS containing 1% BSA for FACS. All samples were loaded on a BD FACS Melody for flow cytometry cell sorting. After sorting, CD68-positive cells were used for cell coculture.
Fetal gonadal cells were fixed with fixation buffer (420801, Biolegend) at room temperature in the dark for 20 min and then washed twice with 1X Intracellular Staining Perm Wash Buffer (421002, Biolegend). The cells were incubated with antibody (SST, 1:100, MA5-16987, Invitrogen) at 4°C for 30 min and washed twice with 1X Intracellular Staining Perm Wash Buffer. Then, the cells were incubated with Alexa Fluor 647-conjugated donkey anti-rat IgG antibodies (1:500, ab150155, Abcam) at 4°C in the dark for 30 min, washed twice and resuspended with 1X Intracellular Staining Perm Wash Buffer. All samples were loaded on a BD C6 Plus for flow cytometry analysis.
Fetal gonadal cells were fixed with fixation buffer (420801, Biolegend) at room temperature in the dark for 20 min and then washed twice with 1X Intracellular Staining Perm Wash Buffer (421002, Biolegend). The cells were incubated with antibody (SST, 1:100, MA5-16987, Invitrogen) at 4°C for 30 min and washed twice with 1X Intracellular Staining Perm Wash Buffer. Then, the cells were incubated with Alexa Fluor 647-conjugated donkey anti-rat IgG antibodies (1:500, ab150155, Abcam) at 4°C in the dark for 30 min, washed twice and resuspended with 1X Intracellular Staining Perm Wash Buffer. All samples were loaded on a BD C6 Plus for flow cytometry analysis.
3
Influenza A Virus Immunostaining in Tissue
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Formaldehyde-fixed heads were decalcified in Osteosoft at room temperature on a roller shaker for 96 hr. Samples were then incubated in 15% sucrose at 4°C for 4 hr, followed by incubation in 30% sucrose overnight. Samples were embedded with Tissue-Tek O.C.T. compound and stored at −80°C until use. 4 µm sections were prepared using a cryotome, dried overnight at 37°C and permeabilized for 5 min with 0.5% Triton-X in PBS. Blocking was performed with 10% donkey normal serum in PBS for 45 min. Primary antibodies were goat anti-influenza A (Purified, AbD Serotec (OBT1551)), rabbit anti-MX1 (anti-AP5 peptide [Meier et al., 1988 (link)]), rat anti-EpCAM (Purified, BD Biosciences (552370)) and mouse anti-E-Cadherin A647 (Purified, BD Biosciences (560062)). Secondary antibodies were donkey anti-goat A488 (Purified, Jackson Immuno Research (705-545-147)), donkey anti-rat A647 (Purified, Abcam (ab150155)) and donkey anti-rabbit A555 (Purified, Invitrogen (A-31572)). Nuclei were stained with DAPI and slides were mounted with FluorSave reagent.
4
Immunofluorescence Protocol for Vascular and Adipose Tissue
5
Immunofluorescence Staining of Vascular and Adipose Tissues
6
Immunofluorescence Staining of Cerebral Organoids
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Cerebral organoids were fixed with 4% paraformaldehyde in PBS. The samples were embedded in Optimal Cutting Temperature Compound (45,833, Sakura Finetek Japan) and sectioned at a thickness of 10 μm using Leica CM3050 S Cryostat. Organoid sections were permeabilized and blocked with blocking solution (0.05% Triton X-100 and 5% goat serum in PBS). Primary antibodies in blocking solution were then added and incubated at 4 °C for overnight, followed by washing and incubation with secondary antibodies. The following antibodies were used—primary antibodies: rabbit anti-pax6 antibody (ab5790, Abcam), mouse anti-β-tubulin III antibody (ab7751, Abcam), and rat anti-ctip2 antibody (ab18465, Abcam) and secondary antibodies: donkey antirat (ab150155, Abcam), donkey antimouse, (A10036, Invitrogen), and donkey antirabbit, (A21206, Invitrogen). Images were viewed using a confocal microscope (TCS SP8, Leica). The image intensity was adjusted using the ImageJ software (NIH).
7
Multicolor Immunofluorescence Staining of Mouse Tissues
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Manual immunofluorescent (IF) staining was done on mouse brain and skin slides blocked for one hour at room temperature (RT) in a humidity chamber with 5% mouse serum, 5% donkey serum, and 0.3% Triton X-100. Primary antibody solutions were prepared in PBS with 0.3% Triton X-100. Primary antibodies used were specific to F4/80 (Novus Biologicals, #NB800-404), Iba1 (Novus Biologicals, #NB100-1028), and DGKα (Bioss Antibodies, #BS-14294R). Tissue was incubated overnight at 4°C in humidity chamber. Fluorescent antibodies (Abcam) used were donkey anti-rat AF647 (#ab150155), donkey anti-rat AF488 (#ab150153), donkey anti-rabbit AF647 (#ab150075), and donkey anti-goat AF488 (#ab150133). Secondary solutions were prepared in PBS with 0.3% Triton X-100 and incubated on slides for three hours at RT in humidity chamber protected from light. Stained slides were mounted using mounting medium with DAPI (Abcam, #ab104139). Slides were imaged using the Leica Thunder Imager at 10x magnification, and analyzed on the Leica Application Suite X (LAS X) software platform.
8
Immunostaining of Electroporated Brain Sections
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Electroporated brains were harvested at P2, fixed in 4% PFA overnight and thoroughly washed with 1 × PBS. The brains were embedded in 4% low-temperature melting agarose in 1 × PBS and cut into 60 µm thick sections on a Leica VT1000S Vibratome. The free-floating sections were permeabilized and blocked with blocking solution (10% FCS and 1% Triton X-100 in 1 × PBS) for 2 h at room temperature and stained in primary antibodies overnight at 4 °C. Primary antibodies, chicken anti-GFP (GFP-1020, AVES Labs) and rat anti-Ctip2 (ab18465, Abcam), were incubated 1:500 in blocking solution. The next day, sections were washed with 1 × PBS, and incubated in secondary antibodies: Alexa Fluor 488 Donkey anti-chicken (703–545-155, Jackson ImmunoResearch), Alexa Fluor 647 Donkey anti-rat (ab 150,155, Abcam), and Alexa Fluor 568 Donkey Anti-Rabbit IgG H&L (ab175470) 1:500 in blocking solution for 2 h at room temperature. Next, the sections were immersed in DAPI for 20 min, washed thoroughly with 1 × PBS, transferred onto microscopic glass slides, and mounted using Mowiol 4–88. Images were taken on an AxioScope A1 epifluorescence microscope.
9
Immunohistochemical Staining Protocol for Cellular Structures
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4% Paraformaldehyde (PFA). Phosphate Buffered Saline (PBS). Ethanol (50%, 70%, 80%, 95%, 100%). Ethanol: Xylene (2:1, 1:1; 1:2). Xylene (100%). Xylene: Paraffin (2:1, 1:1, 1:2). Paraffin (100%). Paraffin oven. Paraffin cassettes. Rotary Microtome (Leica RM2125 RTS). Superfrost Plus Microscope Slides 15x75x1.0 mm (Fisherbrand, # 12–550 15). TRIS-buffered saline (TBS) plus 0.025% Triton X-100. 1% (Bovine serum albumin) BSA in TBS. Primary Antibodies (Cortactin (ab84208, Abcam), Tks5 (ab118575, Abcam Biotechnology), MMP-14 (ab56307, Abcam). Secondary Antibodies (Donkey F(ab’)2 Anti-Goat IgG H&L (Alexa Fluor 647) (ab150139, Abcam), Donkey F(ab’)2 Anti-Rabbit IgG H&L (Alexa fluoro-568) (ab175694, Abcam), Donkey F(ab’)2 Anti-Mouse IgG H&L 488 (ab150155, Abcam). DAPI (NucBlue Fixed Cell Ready Probes reagent, Life technologies - Catalog# R37606). Distilled water. Harris Hematoxylin solution. Lithium carbonate solution. 5% Eosin solution. Mounting medium (ProLong diamond Antifade Mount, Life technologies, #P36961). Coverslips (22mmX50mm 1.5 oz. cover class, Electron Microscopy Sciences Sciences. Catalog#72204–04. ZEISS ELYRA PS.1 super-resolution microscopy, Quorum Spinning Disk Confocal microscope. Imaging analyzing software: ZEISS ZEN 2.3 and Volocity 6.3.
10
Immunofluorescence Visualization of Cell Receptors
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Medium was replaced with either fresh cell culture medium or HAIR solution, and cells were incubated for 1, 3, 6 or 24 h at 37 °C with 5% CO2. Cells were then washed with PBS and fixed in 4% formaldehyde/PBS at room temperature for 10 min. Cells were again washed with PBS and labelled with primary antibody (anti-Syndecan-4 raised in goat (1:5; RDSAF2918; R&D systems) or anti-Cx43 raised in rabbit (1:2000; C6219; Sigma-Aldrich) in PBS overnight. The next day, excess primary antibody was washed away with PBS before applying the secondary antibody (anti-goat 488 raised in donkey (1:100; ab150155; Abcam) or anti-rabbit 488 raised in goat (1:500; A11034, Thermo Fisher Scientific)) and DAPI (1:1000) in PBS for 1 h at room temperature in a humid box. Coverslips were mounted in anti-fade medium (Citifluor™ AF1), and cells were visualized using confocal microscopy.
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