Culture medium was changed 24 hours after transfection start for Opti-MEM medium with 2.5 μg/mL DiI-LDL (Invitrogen), and internalization was stimulated for 3 hours at 37 °C. Cells were first briefly washed with imaging medium (Opti-MEM without phenol red, containing 30 mmol/L N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid and 0.5 g/L NaHCO3 [pH 7.4])/0.2% bovine serum albumin (Invitrogen) then noninternalized LDL was removed from plasma membrane with acidic wash (imaging medium pH 3.5) for 30 seconds. Cells were then fixed with 4% paraformaldehyde and counterstained for LDLR on plasma membrane (Mouse anti-human-LDLR IgG-C7 [Progen Biotechnik GmbH] and Alexa Fluor 488-conjugated chicken anti-mouse IgG [Invitrogen]) for nuclei (Hoechst [Thermo Scientific]) and cell outlines (Draq5 [Biostatus]).
Alexa fluor 488 conjugated chicken anti mouse igg
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 488-conjugated chicken anti-mouse IgG is a secondary antibody used for fluorescent detection and labeling of mouse immunoglobulin G (IgG) in various immunochemical techniques. The antibody is conjugated with the Alexa Fluor 488 fluorescent dye, which emits green fluorescence upon excitation.
Automatically generated - may contain errors
Lab products found in correlation
Mouse monoclonal anti gfap antibody, by Cell Signaling Technology (2 mentions)
Mouse monoclonal anti iba1 antibody, by Santa Cruz Biotechnology (2 mentions)
Alexa fluor 546 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Dii ldl, by Thermo Fisher Scientific (1 mentions)
Draq5, by Biostatus (1 mentions)
Lsm800 confocal microscope, by Zeiss (1 mentions)
Hoechst 33342 stain, by Thermo Fisher Scientific (1 mentions)
Lsm 510 confocal microscope, by Zeiss (1 mentions)
Confocal fluorescence microscopy, by Zeiss (1 mentions)
Rad51, by Merck Group (1 mentions)
γh2ax, by Merck Group (1 mentions)
Alexa fluor 568 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Axio observer microscope, by Zeiss (1 mentions)
5 protocols using alexa fluor 488 conjugated chicken anti mouse igg
1
Quantitative LDL Internalization Assay
2
Immunohistochemical Profiling of Retinal Responses
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunohistochemical staining of 7 µm sections of full-thickness retinas was performed by immunofluorescence with the following primary antibodies: mouse monoclonal anti-GFAP antibody (1:200; Cell Signaling Technology, Inc., Danvers, MA, USA) and mouse monoclonal anti-Iba1 antibody (1:150; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Seven days after ONC, the eyes were enucleated and fixed in freshly made 4% paraformaldehyde in 0.1 M PBS for 1 h, and then the free retina was harvested and flattened. The tissues were dehydrated and embedded in paraffin, and the retinal cross sections were then obtained. The tissues were blocked with 1% bovine serum albumin in PBS for 1 h at room temperature to prevent nonspecific background staining, and then with the primary antibodies overnight at 4 °C. The sections were washed several times with PBS, incubated with Alexa Fluor® 488-conjugated chicken anti-mouse IgG (1:250; Invitrogen, Carlsbad, CA, USA) or Alexa Fluor® 546-conjugated rabbit anti-goat IgG (1:250; Invitrogen, Carlsbad, CA, USA) for 4 h at 4 °C, and then washed again with PBS. The sections were counterstained with 0.1 µg/mL of Hoechst 33,342 stain in PBS (Invitrogen, Carlsbad, CA, USA). The images were analyzed using a Zeiss LSM 800 confocal microscope (Carl Zeiss, Jena, Germany).
3
Immunohistochemical Profiling of Retinal Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunohistochemical staining of 7-µm wax sections of full-thickness retinas was performed by immunofluorescence with the following primary antibodies: mouse monoclonal anti-GFAP antibody (1:200; Cell Signaling Technology), mouse monoclonal anti-Iba1 antibody (1:150; Santa Cruz Biotechnology), mouse monoclonal anti-HIF-1α (1:200; Cell Signaling Technology), and rabbit monoclonal anti-acetyl-H3 (1:200; Cell Signaling Technology). Tissues were blocked with 1% bovine serum albumin in PBS for 1 h at room temperature to prevent nonspecific background staining, and then with primary antibodies overnight at 4 °C. The sections were washed several times, incubated with Alexa Fluor 488–conjugated chicken anti-mouse IgG (1:250; Invitrogen, Carlsbad California) or Alexa Fluor 546-conjugated rabbit anti-goat IgG (1:250; Invitrogen) for 4 h at 4 °C, and then washed again with PBS. The sections were counterstained with Hoechst 33342/PBS (0.1 µg/mL; Invitrogen). Images were analyzed using a Zeiss LSM 510 confocal microscope (Carl Zeiss, Jena, Germany).
4
Immunocytochemical Analysis of DNA Damage Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunocytochemistry was performed as described previously [26 (link)]. Primary antibodies to RAD51 (Millipore, MA, USA) (1:500 dilution) and γH2AX (Millipore, MA, USA) (1:500 dilution) and secondary antibodies to Alexa Fluor 488-conjugated chicken anti-mouse IgG and Alexa Fluor 568-conjugated goat anti-rabbit IgG (Invitrogen, Carlsbad, CA) (1:100 dilution) were used for analysis. Nuclei were visualized by staining with DAPI. The slides were briefly counterstained and analyzed by confocal fluorescence microscopy (Carl-Zeiss MicroImaging Inc., Oberkochen, Germany). The number of RAD51- and γH2AX-foci was evaluated in a mean of 100 cells.
5
Quantifying Cytosolic FUBP1 after H2O2 Stress
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were seeded on 0.13–0.17 mm thick circular cover glasses in 24-well plates and treated with H2O2 for 10 min when the confluence reached 50%. At one hour after H2O2 treatment, cells were fixed and permeabilized by incubating in 100% methanol at −20 °C for 10 minutes. After blocking with 2% BSA in phosphate saline containing 1% Tween 20, the cells were incubated overnight at 4 °C with FUBP1 antibody (1: 50 dilution, sc-271241; Santa Cruz Biotechnology). Alexa Fluor 488 conjugated Chicken anti-Mouse IgG (Invitrogen, A-21200) was used as secondary antibody at 1:2000 dilution. ProLong™ Gold Antifade Mountant with DAPI (Invitrogen, P36931) was added to the cells before mounting and observation under a fluorescence microscope. Images were captured under a 63× objective with a Zeiss Axio Observer Microscope. NIH Image J (v1.52r) was utilized to quantify total fluorescence per cell versus nuclear green fluorescence. Cytosolic FUBP1 fluorescence intensity was calculated by subtracting total cellular fluorescence with nuclear FUBP1 fluorescence signal.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!