The FA triple staining immunofluorescence method has been previously described [22 (link)]. Briefly, FFPE tumor tissue was cut at 4 microns, placed on positively charged slides, and stained with hematoxylin and eosin. Additional sections for immunofluorescence staining were placed in a 60 °C oven for one hour, cooled, deparaffinized, and rehydrated through xylenes and graded ethanol solutions to water in standard fashion. Antigen retrieval was performed by placing slides in Dako’s TRS antigen retrieval solution (Dako) in a calibrated vegetable steamer (Black and Decker) at 94 °C for 25 min. Slides then were placed on a Dako Autostainer for automated staining. The tissue sections were incubated with a primary antibody cocktail of rabbit polyclonal FANC-D2 antibody at a dilution of 1:1,000 and a monoclonal anti-Ki67 mouse antibody (Dako) at a dilution of 1:150, for one hour at room temperature. Sections then were incubated with a secondary antibody (FITC conjugated to anti-rabbit IgG and Alexa fluor 594 donkey anti-mouse IgG, Invitrogen) at 1:1,000 for one hour at room temperature. The sections were mounted on glass slides using a 4′ 6-diamidino-2-phenylindole (DAPI)-containing embedding medium (Vysis Dapi 1, Abbott Laboratories). The slides were analyzed under a Nikon E-400 fluorescence microscope [22 (link)].
Alexa fluor 594 donkey anti mouse igg
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
Alexa Fluor 594 donkey anti-mouse IgG is a secondary antibody conjugate that binds to mouse immunoglobulin G (IgG) molecules. It is designed for use in immunofluorescence and other antibody-based detection techniques.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 donkey anti rabbit igg, by Thermo Fisher Scientific (43 mentions)
Alexa fluor 594 donkey anti rabbit igg, by Thermo Fisher Scientific (16 mentions)
Alexa fluor 488 donkey anti mouse igg, by Thermo Fisher Scientific (8 mentions)
Alexa fluor 488 donkey anti goat igg, by Thermo Fisher Scientific (5 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (4 mentions)
Lsm 700, by Zeiss (4 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 594 donkey anti goat igg, by Thermo Fisher Scientific (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Axio observer z1, by Zeiss (3 mentions)
Alexa fluor 647 donkey anti goat igg, by Thermo Fisher Scientific (2 mentions)
Mouse anti ha tag, by Merck Group (2 mentions)
Goat anti hpiv3, by Abcam (2 mentions)
Sp5 x confocal microscope, by Leica (2 mentions)
Tcs sp5 2 confocal microscope, by Leica (2 mentions)
Anti β actin a1978, by Merck Group (2 mentions)
Anti lc3, by Cell Signaling Technology (2 mentions)
Anti tubulin lf pa0146a, by AbFrontier (2 mentions)
Anti lamin a c sc 6215, by Santa Cruz Biotechnology (2 mentions)
81 protocols using alexa fluor 594 donkey anti mouse igg
1
Immunofluorescence Staining of FANC-D2 and Ki67
2
Immunofluorescence Staining of HeLa Cells
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HeLa cells were cultured on coverslips in 24-well plates overnight. After transfection or/and infection, cells were harvested at the indicated times. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 for 20min at room temperature. After being blocked with 3% bovine serum albumin (BSA) for 30min, cells were incubated with primary antibodies diluted in 1% BSA at 4°C overnight and secondary antibodies diluted in 1% BSA at room temperature for another 1h. Cells were mounted with Fluoroshield (Sigma) and examined by using a Leica confocal microscope after staining with 1 mg/ml 4’,6-diamidino-2-phenylindole (DAPI) in PBS. The primary antibodies used were as follows: goat anti-TIA-1 (1:200, Santa Cruz), goat anti-HPIV3 (1:1000, Abcam), rabbit anti-TIA-1 (1:500, ABclonal), rabbit anti-G3BP (1:500, ABclonal), rabbit anti-eIF4A (1:500, ABclonal), rabbit anti-eIF4E (1:500, ABclonal), rabbit anti-eIF4G (1:200, CST), rabbit anti-phosphorylated eIF2α (1:200, CST), mouse anti-G3BP (1:500, BD Bioscience), mouse anti-HA tag (1:2000, Sigma), mouse anti-Flag tag (1:1000, Sigma), and mouse anti-Myc tag (1:200, Santa Cruz). The secondary antibodies used were as follows: Alexa Fluor 647 donkey anti-goat IgG (1:1000, Invitrogen), Alexa Fluor 488 donkey anti-rabbit IgG (1:1000, Invitrogen), and Alexa Fluor 594 donkey anti-mouse IgG (1:1000, Invitrogen).
3
Immunofluorescence Imaging of HeLa Cells
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HeLa cells cultured on glass-bottom dishes were washed twice with tris-buffered saline (TBS) and fixed in 4% paraformaldehyde/phosphate buffered saline (PBS) for 20 min at room temperature. Subsequently, the cells were permeabilized with 0.5% Triton X-100 in PBS for 5 min at room temperature. After being washed twice with TBS, cells were blocked in 4% BSA/TBS for 1 h, and probed with the primary antibodies overnight at 4 °C. The cells were then washed five times with TBS, followed by secondary antibody incubation at room temperature for 1 h in the dark. Secondary antibodies used were Alexa Fluor® 594 donkey anti-rabbit IgG (Invitrogen), Alexa Fluor® 594 donkey anti-goat IgG (Invitrogen) and Alexa Fluor® 594 donkey anti-mouse IgG (Invitrogen). After being washed five times with TBS, the cells were stained with DAPI and mounted using VECTASHIELD® Mounting Medium with DAPI (H-1200, VECTOR). Immunofluorescence microscopy was performed on a Leica SP5 X Confocal Microscope (Leica Microsystems, Inc).
4
Immunocytochemistry of Cellular Markers
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For immunocytochemistry, cells were washed twice with PBS and fixed in 4% paraformaldehyde (PFA) for 15 min, followed by permeabilization with 0.5% Triton in PBS for 5 min. Cells were then blocked with 4% BSA in PBS for one hour. After that, cells were incubated with primary antibodies in 4% BSA in PBS overnight at 4 °C. The next day, after five PBS washes, cells were incubated in secondary antibodies diluted in 4% BSA in PBS for one hour. Cells were then washed five times with PBS, and fluorescence images were acquired with a Zeiss LSM 710 confocal microscope (Zeiss International, Oberkochen, Germany). Fluorescence intensity was adjusted with ImageJ software (NIH). The primary antibodies used for immunocytochemistry were: lamin A/C (1:250, Millipore MAB3211), progerin (1:250, Cao et al., 2011 [41 (link)]), ∆Np63 (1:250, Biolegend, San Diego, CA, USA #619001), and K14 (1:500, Invitrogen, Waltham, MA, USA MA5-11599). The secondary antibodies used were: Alexa Fluor 488 donkey anti-rabbit IgG (1:1000, Invitrogen), Alexa Fluor 594 donkey anti-rabbit IgG (1:1000, Invitrogen), Alexa Fluor 488 donkey anti-mouse IgG (1:1000, Invitrogen), and Alexa Fluor 594 donkey anti-mouse IgG (1:1000, Invitrogen).
5
Immunostaining analysis for tissue samples
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Immunostaining analysis for tissues was conducted as described previously (Liu et al., 2014 (link)). Rabbit anti-NPHS2 (Abcam ab50339) and mouse anti-4-HNE (Abcam ab48506) antibodies were obtained. Alexa Fluor™ 488 donkey anti-rabbit IgG (Invitrogen, A21206) and Alexa Fluor™ 594 donkey anti-mouse IgG (Invitrogen, A21203) were used for tissue immunostaining assays. Images were taken under TCS SP5 II confocal microscope (Leica Microsystems). Relative fluorescence intensity was calculated by ImageJ software.
6
Chicken Embryo Urogenital Tissue Histology
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Urogenital tissues were dissected from chicken embryos and briefly fixed for 15 min in 4 % paraformaldehyde/PBS at room temperature. Tissues were then cryoprotected by immersion in 30 % sucrose/PBS (overnight at 4 °C), infiltrated with OCT embedding compound. Ten micron frozen sections on slides were treated as previously described [63 (link)]. Secondary antibodies were AlexFluor 488 donkey anti-rabbit IgG, 1:1000, and AlexaFluor 594 donkey anti-mouse IgG, at 1:1500 from Invitrogen). Sections were mounted in Fluorosave (Calbiochem). Images were taken on a Zeiss Axiovision M1.
7
Antibody and Chemical Reagents for Cell Analysis
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The following antibodies were used: anti-Flag (F3165) and anti-β-actin (A1978) (Sigma-Aldrich, St. Louis, MO, USA); anti-GFP (sc-9996) and anti-Lamin A/C (sc-6215) (Santa Cruz biotechnology, Dallas, TX, USA); anti-Tubulin (LF-PA0146A) (AbFrontier, Seoul, Korea); anti-PHF20 (#3934), anti-WDR5 (#13105), and anti-LC3 (#2775) (Cell Signaling Technology, Danvers, MA, USA); anti-HA (#MMS-101R) (Covance, Princeton, NJ, USA); Alexa Fluor 488 donkey anti-rabbit IgG (A21206) and Alexa Fluor 594 donkey anti-mouse IgG (A21203) (Invitrogen, Waltham, MA, USA). The following chemicals were used: hygromycin (H3274), puromycin (P8833), and CQ (C6628) (Sigma-Aldrich, St. Louis, MO, USA); Bafilomycin A1 (#11038) (Cayman, Ann Arbor, MI, USA); and rapamycin (R-5000) (LC laboratories, Woburn, MA, USA).
8
Immunofluorescence Assay for Protein Localization
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For immunofluorescence assay, cells were grown in coverslips and transfected with siRNA or indicated plasmids. After 36–48 h transfection, cells were washed with PBS and fixed with 4% formaldehyde. The embryos then blocked with blocking solution (3% BSA, 0.3% Triton X-100 in PBS) for 30 min, and incubated with primary antibodies, including rabbit anti-mTOR (Cell Signaling Technology, 2983, 1:300), mouse anti-DSTYK (Santa Cruz, sc-374487, 1:100), mouse anti-LAMP1 (Santa Cruz, sc-20011, 1:100), rabbit anti-V5 (Proteintech, 14440-1-AP, 1:200), rabbit anti-RAB7A (Proteintech, 55469-1-AP, 1:200), rabbit anti-DSTYK (Proteintech, 20102-1-AP, 1:200), rabbit anti-TFEB (Proteintech, 13372-1-AP, 1:200), mouse anti-V5 (Abcam, ab27671, 1:300), overnight at 4 °C. Alexa Fluor 488 donkey anti-rabbit IgG (Invitrogen, A21206 , 1:500), Alexa Fluor 568 donkey anti-rabbit IgG (Invitrogen, A10042 , 1:500) and Alexa Fluor 594 donkey anti-mouse IgG (Invitrogen, A21203 , 1:500) were used as a secondary antibody. Immunofluorescence staining was imaged with LSM880NLO confocal microscope with a ×63 oil objective (Carl Zeiss).
9
Histological Analysis of Vascular Grafts
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The grafts were explanted, rinsed with physiological saline and cut into two halves from the middle. The pieces were observed by stereomicroscopy and SEM, after then the explants were cryosectioned to 7 μm in thickness for frozen cross-section on a freezing microtome after fixed and embedded in optimal cutting temperature. The sections were stained with hematoxylin and eosin (H&E) and observed with an inverted microscope as described in our previous study [28 ]. To observe ECs, rabbit anti-von Willebrand factor (vWF, 1: 200, Dako, USA) was performed as primary antibody. To visualize SMCs, mouse α-SMA antibody (α-SMA, 1: 100, Boster, China) was used as primary antibody. Inflammatory cells were stained by CD68 antibody (CD68, 1:200, Abcam, USA), and M2 macrophages were stained by goat anti-human CD206 (CD206, 1:200, Santa Cruz, USA). Alexa Fluor 555 goat anti-rabbit IgG, Alexa Fluor 488 goat anti-mouse IgG (1:200, Invitrogen, USA), Alexa Fluor-594 donkey anti-mouse IgG (1:200, Invitrogen, USA) and Alexa Fluor 488 donkey anti-goat IgG (1:200, Invitrogen, USA) were used as the secondary antibodies, respectively. Sections without incubation with the primary antibody were used as negative controls. Images were observed by the fluorescence microscope (TE2000-U, Nikon Eclipse, Kanagawa, Japan).
10
Antibody Dilutions for Protein Analysis
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α-syn mouse monoclonal syn303 antibody was a generous gift from Dr. V. M. Lee (dilutions: 1:200 for immunofluorescence and 1:2000 for Western blotting). Mouse monoclonal Anti-alpha Synuclein antibody [4D6] (dilution: 1:2000 for Western blotting), rabbit monoclonal [EP1536Y] to alpha synuclein (phospho S129) (dilution: 1:250 for immunofluorescence), and mouse monoclonal antibody [6C5] to GAPDH (dilution: 1:5000 for Western blotting) were purchased from Abcam. Rabbit polyclonal Anti-p62 (SQSTM1) was purchased from MBL. Mouse monoclonal anti- β -III tubulin was from R&D Systems (MAB1195, dilutions: 1:100 for immunofluorescence), β Tubulin antibody (H-235) was from Santa Cruz Biotechnology (sc-9104, dilution 1:3000 for Western), rabbit polyclonal LC3B antibody against LC3 was purchased from Cell Signaling. As the secondary antibodies, we used IRDye® 800CW Goat anti-Rabbit IgG (H + L) (926-32211 LI-COR Biosciences), and IRDye® 680RD Goat anti-Mouse IgG (H + L),(926-68070 LI-COR Biosciences) for Western blotting (1:5000 dilution), and Alexa Fluor 488 donkey anti-rabbit IgG (A21206 Invitrogen), Alexa Fluor 488 goat anti-mouse IgG (A11001 Invitrogen), Alexa Fluor 594 donkey anti-mouse IgG (A21203, Invitrogen), Alexa Fluor 594 donkey Anti-rabbit IgG (A21207, Invitrogen) for immunofluorescence (1:1000 dilution).
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