Snap frozen tissues from Ercc1−/Δ and WT mice were lysed in RIPA lysis and extraction buffer (Thermo Fisher). Protein concentration was determined using BCA protein assay kit (Thermo Fisher). Equal amounts of protein were loaded onto SDS‐PAGE polyacrylamide gels then transferred to 0.2 µm pore size nitrocellulose membranes (Bio‐Rad). Membranes were blocked with 5% BSA in PBS‐Tween for 1 h at room temperature then incubated with primary antibodies overnight at 4℃. Membranes were then probed with secondary antibodies at room temperature. Protein expression was measured by fluorescence using iBright™ FL1000 Imaging System. The density of each blot was quantified by using ImageJ (NIH) normalized to GAPDH. The following primary antibodies were used in this study: rabbit anti‐GAPDH (Cell Signaling Technology, 2118), rabbit anti‐p16 (Santa Cruz, sc‐1207), rabbit anti‐p21 (Abcam, ab7960), rabbit anti‐p‐p65 (Cell Signaling Technology, 3033), mouse anti‐p‐p65 (Cell Signaling Technology, 6956), rabbit anti‐Pai‐1 (Santa Cruz, sc‐8979), rabbit anti‐COX2 (Cell Signaling Technology, 12282). The following secondary antibodies were used in this study: goat anti‐rabbit IgG (H+L) Alexa Fluor Plus 488 (Thermo Fisher, A32731), goat anti‐mouse IgG (H+L) Alexa Fluor 633 (Thermo Fisher, A21052).
Goat anti rabbit igg h l alexa fluor plus 488
Manufactured by Thermo Fisher Scientific
Sourced in United States
Goat anti‐rabbit IgG (H+L) Alexa Fluor Plus 488 is a secondary antibody conjugate designed for use in immunofluorescence applications. It is a goat-derived antibody that specifically binds to the heavy and light chains of rabbit immunoglobulin G (IgG). The antibody is conjugated to the Alexa Fluor Plus 488 fluorescent dye, which can be excited by a 488 nm light source and emits green fluorescence.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (2 mentions)
Goat anti mouse igg h l alexa fluor plus 488, by Thermo Fisher Scientific (2 mentions)
Goat anti mouse igg h l alexa fluor plus 555, by Thermo Fisher Scientific (2 mentions)
Rabbit anti p21, by Abcam (1 mentions)
Rabbit anti p16, by Santa Cruz Biotechnology (1 mentions)
Mouse anti p p65, by Cell Signaling Technology (1 mentions)
Alexa fluor 633 goat anti mouse igg h l, by Thermo Fisher Scientific (1 mentions)
Rabbit anti p p65, by Cell Signaling Technology (1 mentions)
Rabbit anti cox 2, by Cell Signaling Technology (1 mentions)
Ripa lysis and extraction buffer, by Thermo Fisher Scientific (1 mentions)
Rabbit anti gapdh, by Cell Signaling Technology (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Fluorescence microscope, by Leica (1 mentions)
Triton x 100, by Beyotime (1 mentions)
Axiovert 200m, by Zeiss (1 mentions)
Evos fl auto 2, by Thermo Fisher Scientific (1 mentions)
Ca 125, by Santa Cruz Biotechnology (1 mentions)
Fluorescence microscopy, by Zeiss (1 mentions)
8 protocols using goat anti rabbit igg h l alexa fluor plus 488
1
Western Blot Analysis of Cellular Senescence
2
Evaluating Genotoxicity of Colibactin
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The expression of γ-H2AX in bEnd.3 cells was determined to assess the genotoxicity induced by colibactin [6 (link)]. The bEnd.3 cells were infected with APEC XM, APEC XMΔclbA, or APEC XMΔclbA/pclbA for 4 h. Cells were washed three times with PBS and incubated in Dulbecco’s minimal Eagle medium (DMEM; Gibco, USA) with 10% fetal bovine serum (FBS; Gibco, USA) containing gentamicin (100 μg/mL). Then, the expression of γ-H2AX was detected immediately (at 0 h) or at 72 h post-incubation. The cells were washed three times and fixed with 4% paraformaldehyde for 20 min and then permeabilized with 0.1% Triton X-100 for 20 min and processed for immunofluorescence following a standard protocol [19 (link)] using the primary antibody (monoclonal rabbit anti phosphorylated H2AX, Cell Signaling Technology, MA, USA) and the secondary antibody (goat-anti-rabbit IgG (H+L) Alexa Fluor Plus 488, Thermo Fisher Scientific, CA, USA). Then, the cells were stained with 4’, 6-diamidino-2-phenylindole (DAPI, Beyotime Biotechnology, Shanghai, China). Finally, the coverslips were fixed using a fluorescence mounting medium. The GFP fluorescence was detected and photographed by a fluorescence microscope (Leica, Weztlar, Germany).
3
Immunostaining of Nestin-Positive Cells
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For the detection of nestin-positive cells, cultured cells were fixed using 4% paraformaldehyde for 30 minutes (BBI Life Sciences, Shanghai, China; Cat# E672002-0500). After three rinses with PBS, the cells were permeabilized with 0.3% Triton X-100 (Beyotime; Cat# P0096) in PBS for 20 minutes, followed by three PBS washes. We then used blocking solution (5% bovine serum albumin; Odyssey, Xuzhou, China; Cat# 927-40000) to block the cells for 30 minutes at 20°C. Next, cells were incubated at 4°C overnight with monoclonal anti-nestin antibody (rabbit anti-mouse; 1:300; Thermo Fisher Scientific, Waltham, MA, USA; Cat# 14-5843-82), which is a marker of reactive astrocytes (Yang et al., 2006). After three 5-minute washes with PBS, the cells were incubated with secondary antibody (goat anti-rabbit IgG H&L (Alexa Fluor Plus 488); 1:200; Thermo Fisher Scientific; Cat# A32723) at 37°C for 1 hour, followed by 4′,6-diamidino-2-phenylindole (DAPI; 1:100; Beyotime; Cat# C1002) to stain the nuclei. Stained cells were visualized using a fluorescence microscope (Zeiss Axiovert 200M; Zeiss, Heidenheim, Germany).
4
Immunofluorescence Characterization of HGOC Organoids
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The organoids were characterized by immunofluorescence following the protocol of Dekkers et al. (Dekkers et al., 2019 (link)). The following primary antibodies were used to characterize HGOC patients-derived organoids culture in static and passive flow conditions: PAX8 (ProteinTech Group, Germany, EU), WT1 (Abcam, U.K.), and CA-125 (Santacruz Biotechnology, TX). The secondary antibodies that were used are Goat anti-Rabbit IgG (H + L) Alexa Fluor™ Plus 488 (Thermo Fisher Scientific Waltham, Massachusetts, United States) and Goat anti-Mouse IgG (H + L) Alexa Fluor™ Plus 488 (Thermo Fisher Scientific Waltham, Massachusetts, United States). The images were acquired with an EVOS FL Auto 2 (Thermo Fisher Scientific Waltham, Massachusetts, United States).
5
Immunofluorescence Localization of Actin and Flag
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Forty-eight hours after transfection, cells were seeded on glass coverslips and then fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100, and blocked for 1 h in 2% BSA. Immunostaining was conducted with mouse anti-actin antibody (1 : 1000, ProteinTech) and rabbit anti-Flag antibody (1 : 200, ProteinTech) overnight at 4°C. Goat anti-mouse IgG (H+L) Alexa Fluor Plus 555 and goat anti-rabbit IgG (H+L) Alexa Fluor Plus 488 secondary antibodies (Invitrogen, USA) were used at 1 : 1000 at room temperature for 1 h. The cell nuclei were stained with DAPI (6-diamidino-2-phenylindole). Protein localization was observed by fluorescence microscopy (Carl Zeiss, Germany).
6
Assessing Proliferation in Porcine OS Cells
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Porcine OS cells (YAP1−/−/flTP53R167H and flTP53R167H/GFP control) were plated on 6 well plates, cultivated till 80% confluency. For fixation, the cells were washed twice with PBS and incubated for 15 min at room temperature in Fixative. Afterward, the cells were washed two times with TBST, permeabilised for 20 min at room temperature with permeabilisation buffer, and blocked for 60 min with 5% BSA. The primary Ki67 antibody (diluted 1:200, MA5-14520, Invitrogen, Waltham, CA, USA) was incubated at 4 °C overnight, afterwards the secondary antibody (Goat Anti-rabbit IgG (H+L) Alexa Fluor Plus 488, diluted 1:300; A32731, Invitrogen) was added and incubated for 60 min at room temperature. 300 nM DAPI (D9564, Sigma) was incubated for 10 min at room temperature (protected from the light). The signal detection was performed under a fluorescence microscope.
7
Immunofluorescence Analysis of USP12 and YAP in AGS Cells
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The AGS cells were fixed with 4% paraformaldehyde for 10 min and permeabilized 0.2% Triton X-100 (T8200, Solarbio) for 10 min at room temperature. Subsequently, the cells were blocked with PBS supplemented with 5% BSA (ST025, Beyotime) for 1 h. AGS cells were incubated overnight at 4 °C with rabbit anti-USP12 polyclonal antibody (PA5-139844, Invitrogen) and mouse anti-YAP antibody (SC-110199, Santa Cruz). Then the following day, cells were incubated at room temperature for 1 h using Goat anti-Mouse IgG (H + L) Alexa Fluor™ Plus 555 (Invitrogen, 32727) for mouse antibody and Goat anti-Rabbit IgG (H + L) Alexa Fluor™ Plus 488 (Invitrogen, 32731) for rabbit antibody. Three washes later, the cells were stained with DAPI (Beyotime, C1005) to visualize the cell nucleus. The images were captured with a confocal laser scanner and further processed and analyzed using ImageJ.
8
Immunofluorescent Staining of Neural Markers
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After the detection of FISH chromogenic reaction, sections were well washed in 1 × PBS and incubated at RT for 1 h with blocking serum (normal goat serum 1:5 in 1 × PBS containing 0.1% Triton X-100 from Sigma, St. Louis, MO, USA) and subsequently with primary antisera against different neural markers (Table 3 ), ON at 4 °C. Sections were then well washed in 1 × PBS and incubated with secondary antibodies: goat anti-rabbit IgG (H+L) Alexa fluor™ Plus 488 (Invitrogen by Thermo Fisher Scientific, Waltham, MA, USA, ref A32731), goat anti-mouse IgG (H+L) Alexa fluor™ Plus 488 (Invitrogen by Thermo Fisher Scientific, ref A32723), 1:1000 in PBS/DEPC, and Alexa Fluor 488–conjugated Streptavidin, 1:500 (Jackson Immunoresearch, ref 016–540–084).
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