Several primary antibodies were used: OPA1 (612607; BD Biosciences), PDH (ab110333; Abcam), GAPDH (MA5-15738; Thermo), HSP60 (12165; Cell Signaling), TOM20 (11802-1-AP; Proteintech), MPC1 (14462; Cell Signaling Technology), MPC2 (46141; Cell Signaling Technology), CS (14309; Cell Signaling Technology), ACO2 (6571; Cell Signaling Technology), DRP1 (611113; BD Biosciences), MFF (Gandre-Babbe and van der Bliek, 2008 (link)), IDH2 (56439; Cell Signaling Technology), Fumarase (4567; Cell Signaling Technology), SDHA (11998; Cell Signaling Technology), SDHB (ab14714; Abcam), SDHC (14575-1-AP; Proteintech), SDHD (6847; ProSci), ubiquitin (3933S; Cell Signaling Technology), and p62 (GP62-C; Progen). Secondary antibodies were purchased from Invitrogen: Alexa 488 anti-Rabbit IgG (A21206), Alexa 488 anti-Mouse IgG (A21202), Alexa 568 anti-mouse IgG (A10037), and Alexa 647 anti-mouse IgG (A31571).
Alexa 488 anti mouse igg
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 488 anti-mouse IgG is a fluorescently labeled secondary antibody used to detect and visualize mouse primary antibodies in various immunological and cell biology applications. It is conjugated with the Alexa Fluor 488 dye, which has an excitation maximum at 495 nm and an emission maximum at 519 nm, allowing for detection and analysis using standard green fluorescence detection methods.
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Lab products found in correlation
Alexa 594 anti rabbit igg, by Thermo Fisher Scientific (7 mentions)
Alexa 568 anti mouse igg, by Thermo Fisher Scientific (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Tcs spii, by Leica (3 mentions)
Ubiquitin, by Cell Signaling Technology (2 mentions)
Hsp60, by Cell Signaling Technology (2 mentions)
Gapdh, by Thermo Fisher Scientific (2 mentions)
Epifluorescent microscope, by Zeiss (2 mentions)
Monoclonal anti βiii tubulin antibody, by Merck Group (2 mentions)
Axiovision image capture software, by Zeiss (2 mentions)
Axiocam hrc, by Zeiss (2 mentions)
Triton x 100, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Iba1 antibody, by Fujifilm (2 mentions)
Micro slides superfrost plus glass slides, by Electron Microscopy Sciences (2 mentions)
Cy3 anti rabbit igg, by Jackson ImmunoResearch (2 mentions)
Fluoromount g, by Electron Microscopy Sciences (2 mentions)
Monoclonal anti aβ 6e10, by Fortrea (2 mentions)
Tom20 11802 1 ap, by Proteintech (1 mentions)
29 protocols using alexa 488 anti mouse igg
1
Comprehensive Mitochondrial Protein Analysis
2
Immunostaining of BBB and Microglia Cells
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Both HBMEC and N9 cells were grown in 24-well plates with coated coverslips and immunostaining performed as already described62 , 64 (link). Briefly, HBMEC coverslips were incubated overnight at 4 °C with primary antibodies anti-P-gp (1:50, Calbiochem), anti-MRP1 (1:100, Millipore) and anti-BCRP (1:100, Millipore) and N9 cells coverslips were incubated overnight at 4 °C with rabbit polyclonal anti-NF-κB p65 (C-20) (1:200, Santa Cruz Biotechnology). Incubation with secondary antibodies Alexa 594 anti-rabbit IgG (1:500) and Alexa 488 anti-mouse IgG (1:500) (Invitrogen) lasted for 2 h at room temperature. Nuclei were counterstained with DAPI. Between incubations cells were washed three times with PBS. HBMEC staining was examined using a Leica DFC 490 camera (Leica, Germany) adapted to an AxioScope.A1 microscope (Zeiss, Germany), ZEN 2012 blue edition software by Carl Zeiss Microscopy GmbH, 2011. Confocal fluorescent Z-series N9 cells were acquired using on a Leica SP5 live upright confocal (Leica, Wetzlar), using a 63 × 1.3NA oil immersion objective, the UV lamp and DPSS 561 nm yellow-green laser. Post-acquiring treatment was performed using ImageJ software (NIH, USA).
3
Immunocytochemical Localization of RGC
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Fixed cells were washed in several changes of phosphate buffered saline (PBS) before permeablisation and blocking in PBS containing 3% bovine serum albumin (BSA) and 0.1% Triton X-100 (both from Sigma, Poole, UK). Cells were then incubated with monoclonal anti-βIII-tubulin antibody (Cat No. T8660; 1:200 dilution; Sigma) for 1 h at room temperature (RT) and used to localise RGC and their neurites. Cells were then washed in several changes of PBS and incubated with Alexa 488 anti-mouse IgG (Cat No. A-11001; 1:400 dilution; Invitrogen) for 1 h at RT, washed in several changes of PBS and mounted using Vectashield containing DAPI (Vector Laboratories, Peterborough, UK). Slides were then anonymised by a second investigator and viewed using a Zeiss epi-fluorescent microscope equipped with an AxioCam HRc and Axiovision Image capture software (all from Zeiss, Hertfordshire, UK). Immunocytochemistry included controls with primary antibody omitted, that were used to set background levels of nonspecific staining (not shown) prior to image capture.
4
Investigating TGFβ1 Signaling Pathways
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TGFβ1 was a kind gift from Dr F.W. Ruscetti (Biological Response Modifiers Program, FRCF, NCI, Maryland, USA). All fluorophore-coupled reagents (Alexa546-phalloïdin, Alexa488 anti-mouse IgG) were purchased from Invitrogen (Carlsbad, CA, USA). HDAC6 inhibitors (tubacin and CAY10603), SMAD3 inhibitor SIS3 and TGFBR inhibitor Galunisertib (LY2157299) were from Selleckchem (Houston, TX, USA). Cell nucleus stain 4′,6′-diamidino-2-phenylindole (DAPI) was from Invitrogen (Carlsbad, CA, USA). Anti-SMAD2, -SMAD3 and phosphoSMAD2(Ser465/467) and all HRP-coupled secondary antibodies were from Cell Signaling Technology (Boston, MA, USA). Anti-phosphoSMAD3(Ser423/425) was from Abcam (Cambridge, MA, USA). Anti-tubulin and anti-acetylated tubulin were from Sigma Aldrich (Oakville, ON, CA). Anti-SARA and anti-EEA1 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit TrueBlot was purchased from Rockland Immunochemicals (Limerick, PA, USA). Pimonidazole hydrochloride and anti-pimonidazole mouse antibody were purchased from Hypoxyprobe (Burlington, MA). ITGB2 blocking antibody was from Novus Biologicals (Centennial, CO, USA). Anti-DDK antibody was from Origene.
5
Tissue Analysis of Tumor and Renal Samples
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Tumor and renal fixed tissue sections (5 μ) were obtained to analyze tissue structure and fibrosis using classical hematoxylin-eosin and Masson staining, as well as to analyze the degree of angiogenesis, fibrogenesis and cell proliferation using the expression of CD31, α-smooth muscle actin (alpha-sma) and Ki67 by immunohistochemistry, respectively. Sections were deparaffinised, hydrated through graded ethanol steps, briefly rinsed in water and blocked at room temperature using TBSA-BSAT (10 mM Tris, 0.9% NaCl, 0.02% sodium azide, 2% bovine serum albumin and 0.1% Triton-x100 detergent). Slices were incubated overnight at room temperature with primary antibodies against 1:50 CD31 (Abcam, ab28364), 1:250 alpha-sma (Abcam, ab5694) and 1:500 Ki67 (DAKO, IR626) followed by incubation with the corresponding secondary antibodies either Alexa 488 Anti-rabbitt IgG (Invitrogen, A11008) or Alexa 488 Anti-mouse IgG (Invitrogen, A11001) for 5 hours diluted in TBSA-BSAT (1:500). Nuclear staining was performed using DRAQ-5th (Red Fluorescen Cell-Permeable DNA probe, Biostatus Limited, United Kingdom). Immunofluorescence analysis was performed using Olympus BX61 microscope. Fluorescence quantification was performed using Leica Application Suite Advanced Fluorescence software and ImageJ software.
6
Immunofluorescent Analysis of Intestinal Colonoids
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Whole colonoids were collected by gently dissolving Matrigel in ice-cold PBS and fixed for 30 min at room temperature in 4% paraformaldehyde (PFA, Sigma). Colonoids were then transferred to organoid washing buffer (PBS containing 0.1% Triton X-100 and 0.2% BSA), then distributed into a 24-well plate. For immunofluorescent staining, colonoids were permeabilized and blocked in PBS containing 0.5% Triton X-100 and 1% BSA (Sigma) for 1 h at room temperature, then incubated in blocking buffer containing primary antibody overnight at 4°C. Primary antibodies used were Chromogranin A (Santa Cruz #Sc-1488) and phospho-histone H2A.X (Ser139; Cell Signaling Tech #2577). Colonoids were incubated with corresponding secondary antibody Alexa 488 anti-mouse IgG (Invitrogen #A11029), in blocking buffer for overnight at 4°C, with 1ug/ul DAPI added for the final 15 mins of incubation. Colonoids were washed 4−5 times (2 h each), then cleared in fructose-glycerol clearing buffer (60% (vol/vol) glycerol and 2.5 M fructose) for 15 mins before imaging on a Zeiss LSM 780 confocal microscope [27] (link). Image analysis was performed using Zen and Fiji and/or ImageJ software.
7
Mitochondrial Dynamics and Protein Quantification
8
SARS-CoV-2 and Influenza A Infection Assay
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SARS-CoV-2 (JPN/TY/WK-521) was obtained from the National Institute of Infectious Diseases, Japan. Influenza A (A/Puerto Rico/8/34 (H1N1), PR8) was kindly provided by Dr. Hideki Hasegawa (National Institute of Infectious Diseases). Human umbilical vein endothelial cells (HUVECs) were obtained from LIFELINE Cell Technology (#FC-0003). Anti-Spike antibody (SA39) was generated by Cell Engineering Corporation (Osaka, JAPAN). Antibodies for p21 (#2947), γH2AX (#9718), Rab5a (#46449), and Tissue factor (#55147) were obtained from Cell Signaling Technology. Antibody for GAPDH (#ab105428) was obtained from Abcam. Antibody for Ki-67 (#M7240) was obtained from Dako. Alexa594-anti-rabbit IgG (#A21207), Alexa488-anti-mouse IgG (#A11029), Alexa594-anti-mouse IgG (#A11005) were obtained from Invitrogen.
9
Whole-Mount In Situ Hybridization and Antibody Staining in Zebrafish
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Whole-mount in situ hybridization was performed as described previously (Yamamoto et al., 2010 (link)). The zebrafish mib2 probe was generated from a pCR TOPOII vector plasmid in which the mib2 cDNA fragment was subcloned. All probes have been previously published: her4.1 (Takke et al., 1999 (link)), elavl3 (Kim et al., 1996 (link)), xirp2a (Schröter and Oates, 2010 (link)). Whole-mount antibody staining was performed using the following antibodies: anti-Myosin heavy chain (F59, DSHB) and Alexa-488 anti-mouse IgG (Invitrogen).
10
Myosin Heavy Chain Immunostaining
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Differentiated iFDM cells were fixed with 4% paraformaldehyde for 10 min then permeabilized and immunostained using antimyosin heavy chain antibody (MF20, monoclonal supernatant 1/10 dilution, Developmental Studies Hybridoma Bank, U. of Iowa) and detected with Alexa 488 anti-mouse IgG (Invitrogen, Grand Island, NY, USA) and Hoechst 33258 to stain nuclei with methods as described.29 (link)
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