The cells were fixed with 4% paraformaldehyde (Solarbio, Beijing, China) and were blocked with 5% normal goat serum (MultiSciences, Hangzhou, China) in 0.3% Triton-X-PBS and then immunostained for Cox2 (M1 marker, 1:50, Cat. No. sc-514489, Santa Cruz, TX, United States), Ym1/2 (M2 marker, 1:200, Cat. No. ab192029, Abcam, CA, United States), p-p65 (1:100, Cat. No. sc-166748, Santa Cruz, TX, United States), p-p38 (1:100, Cat. No. sc-166182, Santa Cruz, TX, United States), PPARγ (1:50, Cat. No. sc-7273, Santa Cruz, TX, United States), β3-Tubulin (Neurite marker, 1:200, Cat. No. sc-80005, Santa Cruz, TX, United States), MAP2 (1:2000, Cat No. ab32454, Abcam, CA, United States). Alexa Fluor 488-conjugated goat anti-rabbit IgG, Alexa Fluor 594-conjugated goat anti-mouse IgG (1:200, Cat No. ab150077 and ab150116, Abcam, CA, United States) were used as secondary antibodies. DAPI (Biomol, Hamburg, Germany) was used for counterstaining. Fluorescence images were captured using a fluorescence microscope system (OLYMPUS IX73, Olympus) at size of 259 × 346 μm. Ten slices of each group were captured. The images were analyzed with ImageJ (NIH) as described previously [28 (link)].
Alexa fluor 594 conjugated goat anti mouse igg
Manufactured by Abcam
Sourced in United States
Alexa Fluor 594-conjugated goat anti-mouse IgG is a secondary antibody conjugated with the Alexa Fluor 594 fluorescent dye. It is designed to detect and visualize mouse immunoglobulin G (IgG) in various immunoassays and imaging applications.
Automatically generated - may contain errors
Lab products found in correlation
Revertaid m mulv reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Anti dykddddk flag antibody, by Fujifilm (2 mentions)
Anti myc tag monoclonal antibody, by Fujifilm (2 mentions)
Alexa fluor 647 conjugated goat anti rabbit igg, by Abcam (2 mentions)
Penicillin streptomycin solution, by Merck Group (2 mentions)
Isogen, by Nippon Gene (2 mentions)
Triton x 100, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Dojindo Laboratories (2 mentions)
Normal goat serum (ngs), by MultiSciences Biotech (1 mentions)
β3 tubulin, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg, by Abcam (1 mentions)
Pparγ, by Santa Cruz Biotechnology (1 mentions)
Paraformaldehyde (pfa), by Solarbio (1 mentions)
Screenfect a, by Fujifilm (1 mentions)
3 4 5 dimethyl 2 thiazolyl 2 5 diphenyl 2h tetrazolium bromide mtt, by Merck Group (1 mentions)
Dna ligation kit, by Takara Bio (1 mentions)
Nh4cl, by Fujifilm (1 mentions)
2 2 dipyridyl, by Fujifilm (1 mentions)
Cocl2, by Fujifilm (1 mentions)
Mg132 z leu leu leu cho, by Peptide Institute (1 mentions)
9 protocols using alexa fluor 594 conjugated goat anti mouse igg
1
Immunostaining of Microglia and Neurons
2
Analyzing Parkin-Mediated Protein Degradation
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NH4Cl, 2,2′-dipyridyl, CoCl2, Dulbecco’s modified Eagle’s medium (DMEM), ScreenFect™ A, an anti-DYKDDDDK (FLAG) antibody, and an anti-Myc tag monoclonal antibody were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Penicillin–streptomycin solution, fetal bovine serum (FBS), geneticin (G418), CHX, bafilomycin A1, and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). MG132 (Z-Leu-Leu-Leu-CHO) was purchased from the Peptide Institute (Osaka, Japan). Isogen was from Nippon Gene (Toyama, Japan), Revert Aid™ M-MuLV Reverse Transcriptase from MBI Fermentas (Vilnius, Lithuania), Go Taq polymerase from Promega (Madison, WI), and KOD Fx Neo from Toyobo (Tokyo, Japan). The DNA Ligation Kit was obtained from Takara Bio Inc. (Shiga, Japan). 4′,6-Diamidino-2-phenylindole (DAPI) was purchased from Dojindo (Kumamoto, Japan). Anti-Parkin and anti-Hsc70 antibodies were from ProteinTech (Rosemont, IL, USA). An anti-ubiquitin antibody (clone FK2) was from StressMarq Bioscience. Alexa Fluor® 594-conjugated goat anti-mouse IgG and Alexa Fluor® 647-conjugated goat anti-rabbit IgG were obtained from Abcam (Carlsbad, CA, USA).
3
Immunofluorescent Labeling of hFOs
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hFOs were sectioned at a thickness of 16 μm. hFOs sections were treated with 1% Triton X-100 for 30 mins. After washing with PBS three times, hFOs sections were blocked with 5% bovine serum albumin (Solarbio, A8020) for 1 h at 37 °C. The hFOs sections were then incubated with primary antibodies overnight at 4 °C. After washing with PBS three times, hFOs sections were incubated with secondary antibodies for 1 h and DAPI (10 μg/ml Invitrogen, D1306) for 5 mins at room temperature. Primary antibodies used in this study were mouse anti-Ki67 (1:1,000, Cell Signaling Technologies, 9449), rabbit anti-FOXG1 (1:100, Abcam, ab18259), rabbit anti-TBR2 (1:800, Cell Signaling Technologies, 81493), mouse anti-SOX2 (1:200, Invitrogen, MA1-014), rabbit anti-PAX6 (1:100, Proteintech, 12323-1-AP), rabbit anti-MAP2 (1:200, Proteintech, 17490-1-AP), mouse anti-GAD67 (1:200, Abcam, ab26116) and mouse anti-DlX2 (1:200, Santa cruz, sc-393879). The secondary antibodies were Alexa Fluor 594-conjugated goat anti-mouse IgG (1:300, Abcam, ab150116), Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:200, Invitrogen, A-11012), Alexa Fluor 488-conjugated goat anti-rabbit (1:200, Invitrogen, A-11008), goat anti-mouse Cy3 (1:200, BOSTER, BA1031) and goat anti-mouse CoraLite488 (1:200, Proteintech, SA00013-1). All antibodies used in this study were also shown in Supplementary Data 4 .
4
Nrf2 Regulation and Cellular Homeostasis
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Dulbecco's modified Eagle's medium, tBHQ, hydrogen peroxide, an anti-DYKDDDDK (FLAG) antibody, anti-Myc tag monoclonal antibody, anti-GFP(VC) antibody (mFX75), and horseradish-peroxidase-conjugated goat anti-mouse IgG were purchased from Wako Pure Chemical Industries. MG132 (Z-Leu-Leu-Leu-H) was purchased from the Peptide Institute. Mithramycin A was from Cayman Chemicals. Penicillin-streptomycin solution, fetal bovine serum, and geneticin (G418) were from Sigma Chemical Co. Nitrocellulose membrane, horseradish-peroxidase-conjugated goat anti-rabbit IgG, and 4-chloro-1-naphthol were purchased from Bio-Rad Laboratories. Isogen was from Nippon Gene, and Revert Aid M-MuLV Reverse Transcriptase was from MBI Fermentas. KOD Plus Neo and Fx Neo DNA polymerase were from Toyobo. DAPI (4′,6-diamidino-2-phenylindole) was from Dojindo. Alexa Fluor 594-conjugated goat anti-mouse IgG and Alexa Fluor 647-conjugated goat anti-rabbit IgG were from Abcam. Anti-CUL4, anti-KLF15, and anti-AP2α antibodies were from Santa Cruz Biotechnology. An anti-Sp1 antibody was purchased from Cell Signaling Technology. An anti-ubiquitin antibody (clone FK2) was from StressMarq Bioscience. The anti-Nrf2, anti-Keap1, anti-WDR23, and anti-β-actin antibodies were prepared as described previously (12 (link), 55 (link)).
5
Immunofluorescence Labeling of Neuronal Cytoskeleton
6
Immunohistochemistry and Immunofluorescence Protocols
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For immunohistochemistry (IHC), the monoclonal antibodies (mAbs) were anti-CXCL17 (MAB4207, clone 422208; R&D Systems, Minneapolis, MN, USA), anti-CXCL10 (NB600-1426; Novus, Littleton, CO, USA), anti-CXCL9 (MAB392, clone 49106; R&D Systems) and anti-CEA mAb, clone II-7 (Dako, Glostrup, Denmark). Mouse IgG, ready to use (Dako), served as negative control. Anti-mouse Ig ImmPress enhancement reagents kit was purchased from Vector Laboratories (Burlingame, CA, USA). The substrate used was 3,3′-diaminobenzidine (DAB; Vector Laboratories).
For immunofluorescence, the mAbs were FITC-conjugated anti-epithelial cell mAb BerEP4 (F0860, lot 00059670; Dako) and unconjugated anti-CXCL17 mAb. Alexa Fluor 594-conjugated goat anti-mouse IgG (ab150116, Abcam, Cambridge, MA, USA) was used as secondary antibody. Anti-CEA mAb was used as a positive control for indirect staining, and FITC-conjugated mouse IgG2b (X0959; Dako) was used as a negative control.
For immunofluorescence, the mAbs were FITC-conjugated anti-epithelial cell mAb BerEP4 (F0860, lot 00059670; Dako) and unconjugated anti-CXCL17 mAb. Alexa Fluor 594-conjugated goat anti-mouse IgG (ab150116, Abcam, Cambridge, MA, USA) was used as secondary antibody. Anti-CEA mAb was used as a positive control for indirect staining, and FITC-conjugated mouse IgG2b (X0959; Dako) was used as a negative control.
7
Comprehensive Immunostaining Technique
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Immunostaining in vivo and in vitro was performed as described previously (18 (link), 21 (link)). The samples were stained with primary and secondary antibodies as follows: anti-Rab9 mouse monoclonal antibody (1:100, Abcam, Cat #ab2810, RRID:AB_303323 ), anti-LAMP2 rabbit polyclonal antibody (1:100, Sigma, Cat #L0668, RRID:AB_477154 ), anti-LAMP2 mouse monoclonal antibody (1:100, Abcam, Cat #ab25631, RRID:AB_470709 ), anti-TOMM20 rabbit monoclonal antibody (1:100, Abcam, Cat #ab186734, RRID:AB_2716623 ), anti- LC3B rabbit polyclonal antibody (1:100, Abcam, Cat #ab51520, RRID:AB_881429 ), Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:100, Abcam, Cat #ab150081, RRID:AB_2734747 ), and Alexa Fluor 594-conjugated goat anti-mouse IgG (1:100, Abcam, Cat #ab150120, RRID:AB_2631447 ). Fluorescence images were obtained using an LSM700 confocal laser scanning microscope (Zeiss, Cat #LSM700, RRID:SCR_017377 ). Pearson's correlation coefficient was calculated to quantify the colocalization correlation of the intensity distributions between the two channels, as previously described (22 (link)). We performed independent in-vitro experiments three times.
8
Immunofluorescence Staining of OCT4
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For immunofluorescence staining, 104 cells were washed three times in PBS and seeded onto slides. Slides were fixed with 4% paraformaldehyde for 10 min at room temperature, and subsequently permeabilized with 0.1% Triton X-100 (Sigma Aldrich) for 10 min at room temperature. After blocking with 1% bovine serum albumin, slides were incubated with ready-to-use primary antibody against OCT4 (Maxim) and PBS (as a negative control) at 4 °C overnight. The next day, Alexa Fluor 594-conjugated goat anti-mouse IgG (1:200 dilution, Abcam) was added onto slides for 1 h at room temperature to detect the primary antibody. Slides were then counterstained with DAPI to label the nuclei and imaged using a fluorescence microscope.
9
Immunofluorescence Staining and Cell Morphology Observation
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For Immuno uorescence staining, 10 4 cells were washed three times in PBS and seeded onto slides. Slides were xed with 4% paraformaldehyde for 10 minutes at room temperature, and subsequently permeabilized with 0.1% Triton X-100 (Sigma Aldrich) for 10 minutes at room temperature. After blocking with 1% bovine serum albumin, slides were incubated with ready-to-use primary antibody against OCT4 (Maxim) and PBS (as a negative control) at 4°C overnight. The next day, Alexa Fluor 594-conjugated goat anti-mouse IgG (1:200 dilution, Abcam) was added onto slides for 1 hour at room temperature to detect the primary antibody. Slides were then counterstained with DAPI to label the nuclei and imaged using a uorescence microscope.
Cell Morphology Observation hHFMSCs in the well were monitored and photographed under an inverted microscope. After OCT4 transduction, oating hHFMSCs OCT4 and adherent hHFMSCs OCT4 were observed using an inverted microscope and a uorescence microscope respectively.
Cell Morphology Observation hHFMSCs in the well were monitored and photographed under an inverted microscope. After OCT4 transduction, oating hHFMSCs OCT4 and adherent hHFMSCs OCT4 were observed using an inverted microscope and a uorescence microscope respectively.
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