To prepare tissue samples for immunohistochemistry, tissue samples were fixed with a tissue fixation fluid, placed in a paraffin block, and cut into paraffin sections. Dewaxing was first performed with xylene, and subsequently, the tissue sections were dehydrated with gradient alcohol. Sections were stained with H&E to determine if their morphology changed and next rehydrated and microwaved in a sodium citrate buffer (10 mmol/L, pH 6.0) to restore the antigen. Sections were incubated with 0.3% hydrogen peroxide/PBS for 30 min and then blocked with serum. Subsequently, tissue samples were incubated with a 1:200 dilution of rabbit monoclonal anti-IFITM3 antibody (ab15592, Abcam, Cambridge, MA, USA) at 4 °C overnight. The samples were then washed thrice with PBS for 5 min each and incubated with secondary antibody at 37 °C for 30 min. Next, the sections were stained with diaminobenzidine (DAB) and hematoxylin dye; the excess dye was rinsed with running water and then rehydrated with gradient alcohol, and sealed with neutral resin. Finally, the tissue samples were observed under the microscope, and images were acquired.
Ab15592
Manufactured by Abcam
Sourced in United States
Ab15592 is a mouse monoclonal antibody. It is designed to detect the presence of a specific target protein in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Ab13840, by Abcam (3 mentions)
Image studio lite, by LI COR (2 mentions)
Ab18189, by Abcam (2 mentions)
Imagej, by LI COR (2 mentions)
Cleaved notch1 val1744, by Cell Signaling Technology (2 mentions)
Ps1 ntf, by Merck Group (2 mentions)
Ps1 ctf, by Merck Group (2 mentions)
Ps2 ctf, by Abcam (2 mentions)
Aph 1al, by Thermo Fisher Scientific (2 mentions)
Anti fragilis, by Abcam (2 mentions)
Rab7 b 3, by Santa Cruz Biotechnology (2 mentions)
C myc, by Roche (2 mentions)
Immobilon p pvdf, by Merck Group (2 mentions)
Immobilon fl pvdf, by Merck Group (2 mentions)
β tubulin 3, by Merck Group (2 mentions)
Irdye 800cw goat anti rabbit igg h l, by LI COR (2 mentions)
Sds page gel, by Bio-Rad (2 mentions)
β actin c4, by Santa Cruz Biotechnology (2 mentions)
Ab109429, by Abcam (2 mentions)
N cadherin d4r1h, by Cell Signaling Technology (2 mentions)
11 protocols using ab15592
1
Immunohistochemical Tissue Processing Workflow
2
Immunofluorescence Analysis of Germ Cell Markers
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Formaldehyde-fixed tissue sections, DDX4C25-positive cells and oocytes were incubated with antibodies for the detection of DAZL (ab34139; Abcam), DDX4 (DDX4C25 antibody, ab13840; Abcam and DDX4351 antibody, 17545-1-AP; ProteinTech, UK), DPPA3 (ab19878; Abcam), IFITM3 (ab15592; Abcam) and PRDM1 (PA5-20310; ThermoFisher, UK). All images were acquired using a Leica SP8 microscope with hybrid detectors and x63 oil immersion lens. Fluorochromes were imaged sequentially. Images were then analysed on ImageJ (NIH, USA).
3
Isolation and Purification of Female Germline Stem Cells
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Ovaries were collected from 5-day-old pou5f1/GFP transgenic mice × C57BL/6 F1 hybrid mice. Female germline stem cells (FGSCs) were isolated and purified using a method described elsewhere [16] (link). Briefly, dissected ovarian tissues were incubated in 1 mg/ml collagenase (type IV; Sigma) at 37 °C with gentle agitation for 15–20 min. After washing, ovarian tissues were incubated in 0.05% trypsin and 1 mM EDTA at 37 °C for 5–7 min. Sheep anti-mouse IgG magnetic beads (Dynal Biotech) were incubated with an anti-fragilis antibody (ab15592, Abcam) for 30 min at room temperature. The magnetic bead/antibody mixture was incubated with the isolated cell suspension for another 30 min at room temperature. Then, the mixture of cells and magnetic beads was placed on a magnetic bead separator for 2–3 min, and the supernatant was removed. The fraction on the inner side of the eppendorf tube was collected and rinsed twice with PBS, resuspended in PBS, and further purified by FACS, in accordance with the manufacturers’ instructions. The purified FGSCs were placed in FGSC culture medium and cultured on mitotically inactivated STO feeder cells in 24-well plates at 37 °C with 5% CO2.
4
Western Blotting of Protein Samples
5
Immunostaining of Germ Cell Markers
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Cells were fixed in 4 % paraformaldehyde for 15 min, washed twice in phosphate-buffered saline, and then incubated with 10 % goat serum at 37 °C for 15 min. Primary anti-Fragilis antibody (ab15592, Abcam, 1:200), anti-Mvh antibody (ab13840, Abcam, 1:100) or anti-Prmt5 (ab109451, Abcam, 1:50), and secondary antibody IgG (SA00003-2, Proteintech, 1:200) were used for immunostaining. Images were acquired using a Nikon A1Si confocal microscope or Leika DMI 3000 B inverted microscope.
6
Western Blotting of Protein Samples
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Protein samples were subjected to SDS-PAGE gels (Bio-Rad), electrophoresed, transferred to Immobilon-P PVDF (Millipore) or Immobilon-FL PVDF membranes (Millipore) and Western blotting (WB). The following primary antibodies were used: PS1-NTF (a kind gift of Merck Research Laboratories); PS1-CTF (MAB5232, Millipore); PS2-CTF (1987–1, Epitomics); nicastrin was generated in our laboratory; Aph-1aL (38–3600, Invitrogen); Pen-2 (ab18189, Abcam); human IFITM3 (anti-Fragilis, ab109429, Abcam); mouse IFITM3 (anti-Fragilis, ab15592, Abcam); APP (22C11, MAB348, Millipore); SPP (317) was generated in our laboratory; cleaved Notch1 (Val1744), Cell Signaling Technology and SM320, generated in our lab); c-myc (9E10, Roche Life Science); N-cadherin (D4R1H, #13116, Cell Signaling Technology); Rab7 (B-3, sc-376362, Santa Cruz Biotechnology); EEA1 (Ab2900, Abcam); β-actin (C4, sc-47778, Santa Cruz Biotechnology); β-tubulin III (T8578, Sigma-Aldrich); tubulin (ab56676, Abcam). HRP-conjugated anti-rabbit and mouse secondary antibodies (NA9340V, NXA931V, GE Healthcare) for ECL substrate (Pierce) and IRDye 800CW goat anti-rabbit IgG (H+L) or anti-mouse IgG (H+L) secondary antibodies (925–32211, 925–32210, LI-COR) for Odyssey CLx Imaging (LI-COR) were used. For quantification, ImageJ and Image Studio Lite (LI-COR) were used, respectively (for source gels see Supplementary Figure 1 ).
7
Immunofluorescent Staining of Oocyte Stem Cells
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OSCs were fixed with 4% paraformaldehyde for 15 min at room temperature and then incubated in blocking solution (10% normal goat serum in PBS) for 1 h at room temperature. Following the incubation at 37°C for 1 h with primary antibodies (rabbit polyclonal anti-MVH (1 : 200 dilution, ab13840, Abcam), rabbit polyclonal anti-Fragilis (1 : 500 dilution, ab15592, Abcam)), OSCs were incubated with FITC conjugated secondary antibody (goat anti-rabbit IgG, 1 : 1000 dilution) and then were stained by DAPI for 15 min.
8
Immunofluorescence Labeling of Brain Markers
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The prepared tissue sections were briefly rinsed in 0.1 M phosphate-buffered saline (PBS) and incubated with 0.1% Triton X-100 for 30 min. Subsequently, the sections were blocked with 0.1 M PBS containing 0.03% Triton X-100 and 5% goat serum for 1 h. The primary antibodies were anti-IFITM3 (Abcam, ab15592, 1:100), anti-glial fibrillary acidic protein (GFAP) (Abcam, ab4648, 1:200), anti-neuronal nuclear protein (NeuN) (Abcam, ab104224, 1:500), and ionized calcium-binding adapter molecule 1 (Iba1) (Novus Biologicals, NB-1028ss, 1:100). The tissue sections were incubated for 2 h at room temperature of 24°C, kept overnight at 4°C, and washed three times with PBS for 5 min. They were incubated in the dark with Alexa Fluor 488-conjugated goat anti-rabbit (Abcam, 1:300) or Alexa Fluor 594-conjugated goat anti-mouse (Abcam, 1:300) secondary antibody dilutions for 2 h at room temperature, respectively. They were washed three times with PBS for 5 min. The slides were mounted in glycerol, observed under a fluorescence microscope, and photographed.
9
Immunofluorescence Labeling of Brain Markers
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The prepared tissue sections were briefly rinsed in 0.1 M phosphate-buffered saline (PBS) and incubated with 0.1% Triton X-100 for 30 min. Subsequently, the sections were blocked with 0.1 M PBS containing 0.03% Triton X-100 and 5% goat serum for 1 h. The primary antibodies were anti-IFITM3 (Abcam, ab15592, 1:100), anti-glial fibrillary acidic protein (GFAP) (Abcam, ab4648, 1:200), anti-neuronal nuclear protein (NeuN) (Abcam, ab104224, 1:500), and ionized calcium-binding adapter molecule 1 (Iba1) (Novus Biologicals, NB-1028ss, 1:100). The tissue sections were incubated for 2 h at room temperature of 24°C, kept overnight at 4°C, and washed three times with PBS for 5 min. They were incubated in the dark with Alexa Fluor 488-conjugated goat anti-rabbit (Abcam, 1:300) or Alexa Fluor 594-conjugated goat anti-mouse (Abcam, 1:300) secondary antibody dilutions for 2 h at room temperature, respectively. They were washed three times with PBS for 5 min. The slides were mounted in glycerol, observed under a fluorescence microscope, and photographed.
10
Immunohistochemical Evaluation of IFITM3 Protein
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The prepared tissue was xed with a Tissue xation uid, placed in a para n block, and cut into para n sections. Dewaxing was rst carried out with xylene, and then the tissue sections were dehydrated with gradient alcohol. Sections were stained with H&E to determine if their morphology changed and then rehydrated and microwaved in sodium citrate buffer (10 mmol/L, pH 6.0) to restore the antigen. Sections were incubated with 0.3% hydrogen peroxide/phosphate buffered saline for 30 min and then blocked with serum. Subsequently, tissues were incubated with a 1:200 dilution of rabbit monoclonal antibody IFITM3 (ab15592, Abcam, Cambridge, MA, USA) at 4 C overnight. Wash 3 times with phosphate buffered saline (PBS) every 5 minutes, and drop the secondary antibody at 37 ° C for 30 minutes. Next, the sections were stained with diaminobenzidine (DAB) and hematoxylin dye, then the excess dye was rinsed with running water, then rehydrated with gradient alcohol, and sealed with neutral resin. Finally, observe and take pictures under the microscope.
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