4T1 cells were seeded in 12-well plates until reaching a confluency of approximately 70%. Subsequently, the cells were treated with DMSO (2 μmol/L), Oxa (2 μmol/L), APAP (2 μmol/L), OAP2 (2 μmol/L) for 24 h. After the incubation period, the cells were washed 3 times with cold PBS and fixed with 4% paraformaldehyde for 10 min at room temperature. To permeabilize the cells, 0.2% Triton X-100 in PBS was applied for 5 min at room temperature. After that, the cells were blocked with 5% BSA (Sangon Biotech (Shanghai) Co., Ltd., A500023) in TBST for 0.5 h at room temperature. Primary antibodies were incubated at 4 °C overnight. After washing the cells 4 times with TBST, and the second antibodies were incubated for 1 h at room temperature. Finally, the nucleus was stained with 4,6-diamidino-2-phenylindole (DAPI, Invitrogen, R37606) or Hoechst 33342 (Beyotime Biotechnology, C1025). The antibodies and their working diluted concentrations were as follows: anti-DNA (PROGEN, 1:200), Sam50 (1:500), Bak (1:200), CRT (Abcam, ab215191, 1:500), HMGB1 (Abcam, ab79823, 1:250), Phospho-STING (CST, 62912, 1:400), CoraLite488-conjugated goat anti-rabbit IgG (H + L) (Proteintech, SA00013-2, 1:500), and Cy3-conjugated goat anti-mouse IgG (H + L) (Servicebio, GB21301, 1:500), Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed (Abcam, ab150119, 1:1000).
Cy3 conjugated goat anti mouse igg
Manufactured by Wuhan Servicebio Technology
Sourced in China
Cy3-conjugated goat anti-mouse IgG is a secondary antibody that binds to mouse immunoglobulin G (IgG) molecules. The Cy3 fluorescent dye is attached to the antibody, allowing for detection and visualization of target proteins in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Fitc conjugated goat anti rabbit igg, by Wuhan Servicebio Technology (2 mentions)
Goat serum, by Wuhan Servicebio Technology (2 mentions)
Anti cbs rabbit pab, by Wuhan Servicebio Technology (2 mentions)
Anti c fos mouse pab, by Wuhan Servicebio Technology (2 mentions)
Anti dna, by Abcam (1 mentions)
Coralite488 conjugated goat anti rabbit igg, by Proteintech (1 mentions)
Goat anti mouse igg h l, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Beyotime (1 mentions)
Bovine serum albumin (bsa), by Sangon (1 mentions)
D ap5, by Merck Group (1 mentions)
Pontamine sky blue, by Merck Group (1 mentions)
Tcs sp8 sted, by Leica camera (1 mentions)
L703606, by Merck Group (1 mentions)
Capsazepine, by Merck Group (1 mentions)
Anti gfap, by Wuhan Servicebio Technology (1 mentions)
Antifade mounting medium with dapi, by Beyotime (1 mentions)
Fluoview fv1000, by Olympus (1 mentions)
Ab206655, by Abcam (1 mentions)
Hrp conjugated goat anti rabbit igg, by Wuhan Servicebio Technology (1 mentions)
7 protocols using cy3 conjugated goat anti mouse igg
1
Visualizing Cellular Responses to Treatments
2
Quantification of Hydrogen Sulfide Signaling
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NaHS (2, 4, and 8 nmol), PDTC (2 nmol), D-AP5 (2 nmol), and pontamine sky blue were all purchased from Sigma-Aldrich (St. Louis, MO, United States). NaHS was dissolved in 0.9% normal saline, while the other chemicals were dissolved in dimethyl sulfoxide. Goat serum, anti-CBS rabbit pAb, FITC-conjugated goat anti-rabbit IgG, Cy3 conjugated Goat anti-mouse IgG, and anti-c-Fos mouse pAb were purchased from Servicebio.
3
Localization of EnGAM22 and rEnGAM59 Proteins in Eimeria necatrix
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In order to confirm the localization of EnGAM22 and rEnGAM59 proteins in WFBs of E. necatrix, indirect immunofluorescence assays (IFAs) were performed on purified WFBs and macrogametocytes as described previously [13 (link), 26 (link), 30 (link)]. Briefly, WFBs and macrogametes were placed on 0.1% (v/v) poly-l -lysine-coated glass microscope slides and fixed in methanol (− 20 °C). After blocking overnight in 5% BSA in PBS (BSA/PBS) at 4 °C, the samples were incubated with either rabbit anti-rEnGAM22 pAb (1:100 dilution) and mouse anti-rEnGAM59 pAb (1:100 dilution) or with rabbit anti-rEnGAM22 pAb (1:100 dilution) and mouse anti-WFB pAb (1:100 dilution) for 1 h at 37 °C, washed with 0.03% Tween 20/PBS (PBST) three times for 15 min and incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (1:100 dilution; MultiSciences Hangzhou, China) and Cy3-conjugated goat anti-mouse IgG (1:100 dilution; Servicebio) in BSA/PBS for 1 h at 37 °C, respectively. Before visualization, the samples were rinsed in PBST as described above. Images were obtained using LCM (Leica TCS SP8 STED [STimulated Emission Depletion], Wetzlar, Hessen, Germany). Naïve sera from rabbits and mice were used as a negative control.
4
Hydrogen Sulfide Signaling Pathway Study
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NaHS, L703606, PDTC, Capsazepine, and protamine sky blue were purchased from Sigma-Aldrich (St. Louis, MO, United States). NaHS was dissolved in 0.9% saline, while the other chemicals were dissolved in dimethyl sulfoxide and reconstituted in saline. For the immunohistochemical fluorescence double labeling, the following reagents were purchased from Servicebio (Wuhan, China): Goat serum, anti-CBS rabbit pAb, FITC-conjugated goat anti-rabbit IgG, Cy3-conjugated goat anti-mouse IgG, and anti-c-Fos mouse pAb.
5
Histological Analysis of Mouse Brain Tissue
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The brain tissues were prepared as described above. Isolated mice’s brains were fixed in 4% paraformaldehyde overnight, and then embedded in paraffin. Paraffin blocks were cut into 5 μm-thick sections, which were de-paraffinized and rehydrated after mounting on glass slides. Hematoxylin and eosin (H&E) staining was performed using the H and E dye solution set (Servicebio, Wuhan, China, G1003). Sections were examined using the Digital Slide Scanner (Winmedic, Shandong, China). Immunostaining was performed as following: samples were permeabilized in 0.2% Triton X-100 in TBS for 5 min and were then washed three times in TBS. After blocking in 5% horse serum in TBS at room temperature for 1 h, slides were incubated with anti-GFAP (1:1000, Servicebio, GB12096) overnight at 4 °C, placed in a wet box. GFAP immunome activity was detected with Cy3 conjugated Goat Anti-mouse IgG (1:4000; Servicebio, GB27301). Stained samples were mounted with Antifade Mounting Medium with DAPI (Beyotime, Nantong, China, P0131) and visualized by confocal microscopy (Olympus Fluoview FV1000).
6
Immunofluorescence Staining of Tissue Sections
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For immunofluorescence analysis, formalin-fixed, paraffin-embedded clinical tissue sections were prepared and stained using human CYP27B1, human leukocyte antigen-G (HLA-G), and hCG antibodies and a VECTASTAIN Elite ABC kit (Vector Laboratories) according to the manufacturer's protocol. Rabbit anti-human CYP27B1 monoclonal antibodies (1:500, ab206655, Abcam), mouse anti-human CYP27B1 monoclonal antibodies (1:500, sc-515903, SANTA CRUZ), Mouse anti-human HLA-G monoclonal antibodies (1:50, 4H84, SANTA CRUZ), Rabbit anti-human hCG monoclonal antibodies (1:1000, ab238319, Abcam) were used as the primary antibody. HRP conjugated Goat Anti-Rabbit IgG (1:500, Servicebio), 488 conjugated Goat Anti-Rabbit IgG (1:400, Servicebio), Cy3 conjugated Goat Anti-mouse IgG (1:300, Servicebio) were used as the secondary antibody, all slides were evaluated by two pathologists.
7
Synthesis and Characterization of mPEG-RAFT Agent for Cell Culture Studies
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The chain transfer agent S-1-dodecyl-S-(α,α’-dimethyl-a”-acetic acid)trithiocarbonate conjugated mPEG (mPEG-RAFT, Mw=2000) was provided by Professor Dong Anjie of Tianjin University; All the materials used in the cell culture study were obtained from Sigma-Aldrich (St. Louis, USA). DAPI, anti-fluorescence quenching sealing tablet, chlorpromazine hydrochloride and amiloride hydrochloride were purchased from Solarbio (Beijing, China). Biotinylated HA binding protein was obtained from EMD Millipore (CalBioChem). Alexa Fluor 488-conjugated streptavidin was obtained from Biolegend (San Diego, USA). Anti-collagen I rabbit pAb, anti-α-SMA mAb, anti-CD31 rabbit pAb, CY3 conjugated goat anti-rabbit IgG and CY3 conjugated goat anti-mouse IgG were obtained from Servicebio (Wuhan, China). HepG2 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The male athymic nude mice (nu/nu CD-1, 6 weeks old, 18–22 g) were purchased from Beijing Vital River Laboratory Animal Technologies Co. Ltd (Beijing, China) and maintained at 25°C with free access to food and water. All animal experiments had been approved by the Animal Care Ethics Committee of Qingdao University (Qingdao, China) and were carried out in compliance with the Animal Management Rules of the Ministry of Health of the People’s Republic of China (document no. 55, 2001).
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