Livers from euthanized mice were quickly harvested and embedded in Optimal Cutting Temperature (OCT), and sectioned (6 µm) onto glass slides. Tissues were fixed in 4% paraformaldehyde before immunostaining with ABCC6-specific antibody S-20 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Samples were then incubated at room temperature with Alexa Fluor 488 donkey anti-goat secondary antibody (Invitrogen), followed by DAPI staining of nuclei. Slides were viewed under a Carl Zeiss LSM 510 UV META inverted confocal microscope (Carl Zeiss Microimaging, Thornwood, NY) with a Plan-Apo 63×oil immersion lens using Zeiss AIM 4.2 SP1 software.
Alexa fluor 488 donkey anti goat secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Alexa Fluor 488 donkey anti-goat secondary antibody is a fluorescently labeled antibody used for detection in immunoassays. It binds to and labels goat primary antibodies, allowing for visualization and quantification of target proteins or molecules.
Automatically generated - may contain errors
Lab products found in correlation
Human fc blocking solution, by BioLegend (2 mentions)
Anti cd68 antibody, by Abcam (1 mentions)
Anti siglec 9 antibody, by R&D Systems (1 mentions)
Pdvf membrane, by GE Healthcare (1 mentions)
A11055, by Thermo Fisher Scientific (1 mentions)
Af596, by R&D Systems (1 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (1 mentions)
P0160, by Agilent Technologies (1 mentions)
A9418, by Merck Group (1 mentions)
5 protocols using alexa fluor 488 donkey anti goat secondary antibody
1
Immunostaining of Mouse Liver Sections
2
ACE2 Cell Surface Analysis by Flow Cytometry
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For cell surface analysis of ACE2, we harvested cells and washed them in fluorescence-activated cell sorting (FACS) buffer (2% FBS in 1× PBS). Cells were resuspended in a 1:50 dilution of human FC blocking solution (number 422302; BioLegend) and incubated on ice for 10 min. Human ACE2 antibody or goat IgG isotype control was then added to the cells to obtain the final concentration of 5 μg/ml followed by 1 h of incubation on ice. The cells were washed with FACS buffer and incubated for 30 min on ice in the dark with a 1:400 dilution of Alexa Fluor 488 donkey anti-goat secondary antibody (number A11055; Invitrogen). The cells were washed and resuspended in FACS buffer. Data were collected using a BD LSR II flow cytometer and analyzed with FlowJo software (version 10).
3
Immunofluorescent Staining of Siglec-9 and CD68
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After rehydration in a graded alcohol series, slides of BALF were washed in TBS and heated at 100 °C for 10 min in sodium citrate buffer for epitope retrieval. Slides were incubated with blocking reagent (Dako Japan Ltd., Kyoto, Japan) for 1 h at room temperature, and then were incubated overnight with anti-CD68 antibody (Abcam, Cambridge, UK) plus anti-Siglec-9 antibody (R&D Systems, Minneapolis, USA) or matched isotype controls (Becton Dickinson) at 4 °C. After washing, samples were incubated with Alexa Fluor 594-goat anti-mouse and Alexa Fluor 488-donkey anti-goat secondary antibody (Invitrogen, California, USA) for 60 min at room temperature. Nuclei were counterstained with DAPI. Subsequently, H&E staining were carried out in the slides which were double immunostaining for Siglec-9 and CD68.
4
Adenoviral Transduction of HEK293 Cells
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HEK293 cells were infected with a multiplicity of infection (MOI) of 2 or 5 for 24–48 h with Ad5.CMV.sIL21R and Ad5.CMV.Null or Ad5.CMV.eGFP as negative controls. For immunocytochemistry, anti-IL21R antibody (AF596, R&D Systems, Minneapolis, MN, USA) and Alexa Fluor 488 donkey anti-goat secondary antibody (A11055, Invitrogen, Carlsbad, CA, USA) were used at a dilution of 1:100 and 1:200, respectively. For immunodetection, cell pellet was homogenized in lysis buffer (RIPA) and total protein was determined from lysed cells and supernatants using the Pierce BCA Protein Assay (232,227, Thermo Fisher Scientific, Waltham, MA, USA). Protein extracts and supernatants were loaded onto denaturing acrylamide gels and then electrotransferred to PDVF membranes (10,600,023, GE Healthcare, Chicago, IL, USA). Anti-IL21R primary antibody (AF596, R&D Systems) was used at 1:1000 dilution and goat HRP-anti-IgG secondary antibody (P0160, Dako, Agilent Technologies, Santa Clara, CA, USA) at 1:10,000 were used in the presence of 5% (w/v) of BSA (A9418, Sigma-Aldrich, St. Louis, MO, USA).
5
ACE2 Expression Analysis by Flow Cytometry
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For cell surface analysis of ACE2, we harvested cells and washed them in FACS Buffer (2% FBS in 1X PBS). Cells were resuspended in 1:50 dilution of human FC blocking solution (BioLegend; #422302) and incubated on ice for 10 min. Human ACE2 antibody or goat IgG isotype control was then added to the cells to obtain the final concentration of 5 μg/mL followed by 1h incubation on ice. The cells were washed with FACS buffer and incubated for 30 minutes on ice in the dark with 1:400 dilution of Alexa Fluor 488 donkey anti-goat secondary antibody (Invitrogen; #A11055). The cells were washed and resuspended in FACS buffer. Data were collected using a BD LSR II flow cytometer and analyzed with FlowJo software (version 10).
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