For immunofluorescence (IF), cells were fixed on coverslips and permeabilized with 0.1% Triton-X in PBS at room temperature for 10 min, and blocked with 5% normal goat serum in PBS at room temperature for 10 min. Cells were then incubated with primary antibodies at room temperature for 1 h, followed by incubating with secondary antibodies at room temperature for 1 h. The samples were then processed using the RNA FISH protocol, as described above. Primary antibodies and the dilution used for IF were anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2) (Abcam; ab5095) (1:100), anti-Nanog (Abcam; ab80892) (1:100), and anti-EZH2 (Active Motif; 39933) (1:100). Secondary antibodies and the dilution used for IF were Alexa Fluor® 405 goat anti-rabbit IgG (H+L) (Life Technology; 1575534) (1:100) and Alexa Fluor® 488 F(ab’)2 fragment of goat anti-rabbit IgG (H+L) (Life Technology; 1618692) (1:100).
Alexa fluor 488 f ab 2 fragment of goat anti rabbit igg h l
Manufactured by Thermo Fisher Scientific
Alexa Fluor® 488 F(ab')2 fragment of goat anti-rabbit IgG (H+L) is a fluorescently labeled secondary antibody fragment used for detection and visualization in various immunoassay techniques. It is composed of the F(ab')2 portion of goat-derived antibodies that specifically bind to the heavy and light chains of rabbit immunoglobulins. The Alexa Fluor® 488 dye provides a green fluorescent signal upon excitation.
Automatically generated - may contain errors
Lab products found in correlation
Ab5095, by Abcam (2 mentions)
Ab80892, by Active Motif (2 mentions)
Alexa fluor 405 goat anti rabbit igg h l, by Thermo Fisher Scientific (2 mentions)
Monoclonal anti flag m2, by Merck Group (1 mentions)
Anti ha monoclonal antibody clone ha 7, by Merck Group (1 mentions)
Rabbit polyclonal anti lc3b, by Cell Signaling Technology (1 mentions)
Mouse monoclonal anti β actin, by Merck Group (1 mentions)
Mouse anti phospho histone h2a x ser139, by Merck Group (1 mentions)
4 protocols using alexa fluor 488 f ab 2 fragment of goat anti rabbit igg h l
1
Immunofluorescence and RNA FISH Assay
2
Antibody Detection of Viral and Cellular Proteins
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The primary antibodies used were as follows: mouse monoclonal anti-HSV gB (Virusys), mouse monoclonal anti-HSV1 gC clone 3G9 (Abcam), rabbit polyclonal anti-LC3B (Cell Signaling Technology), mouse monoclonal anti-β-actin, anti-FLAG M2 monoclonal, and anti-HA monoclonal antibody, clone HA-7 (Sigma-Aldrich). For Western blotting, the secondary antibodies used were peroxidase-conjugated AffiniPure F(ab′)2 fragment of goat anti-mouse IgG(H + L) or peroxidase-conjugated AffiniPure F(ab′)2 fragment of goat anti-rabbit IgG(H + L) (Jackson ImmunoResearch). Secondary antibodies for immunofluorescence were AlexaFluor 594 F(ab′)2 fragment of goat anti-mouse IgG(H + L) or AlexaFluor 488 F(ab′)2 fragment of goat anti-rabbit IgG(H + L) (Life Technologies).
3
Immunofluorescence and RNA FISH Assay
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For immunofluorescence (IF), cells were fixed on coverslips and permeabilized with 0.1% Triton-X in PBS at room temperature for 10 min, and blocked with 5% normal goat serum in PBS at room temperature for 10 min. Cells were then incubated with primary antibodies at room temperature for 1 h, followed by incubating with secondary antibodies at room temperature for 1 h. The samples were then processed using the RNA FISH protocol, as described above. Primary antibodies and the dilution used for IF were anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2) (Abcam; ab5095) (1:100), anti-Nanog (Abcam; ab80892) (1:100), and anti-EZH2 (Active Motif; 39933) (1:100). Secondary antibodies and the dilution used for IF were Alexa Fluor® 405 goat anti-rabbit IgG (H+L) (Life Technology; 1575534) (1:100) and Alexa Fluor® 488 F(ab’)2 fragment of goat anti-rabbit IgG (H+L) (Life Technology; 1618692) (1:100).
4
Immunofluorescent Staining Protocols for Histone H2A.X and Cytokeratins
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Mouse anti‐phospho‐histone H2A.X (Ser 139; # 05‐636) was purchase from Millipore (Temecula, CA) and a 1:500 dilution was used for immunofluorescent staining. Rabbit anti‐cytokeratin 8 (CK8; # EP16284) was purchased from Abcam (Cambridge, MA) and a 1:300 dilution was used for immunofluorescent staining.
The CK5 antibody was originally from Covance (Princeton, NJ; Cat. # SIG‐3475) but now is sold by BioLegend (San Diego, CA; Cat. # 905901). 1:1000 dilution was used. Fluorescent secondary antibodies including Alexa Fluor 488 F(ab′) 2 fragment of goat anti‐rabbit IgG (H+L; #A11070) and Alexa Fluor 568 F(ab′) 2 fragment of goat anti‐mouse IgG (H+L; #A11019) were purchase from Life Technologies (Eugene, OR). A 1:500 dilution was for immunofluorescent staining.
The CK5 antibody was originally from Covance (Princeton, NJ; Cat. # SIG‐3475) but now is sold by BioLegend (San Diego, CA; Cat. # 905901). 1:1000 dilution was used. Fluorescent secondary antibodies including Alexa Fluor 488 F(ab′) 2 fragment of goat anti‐rabbit IgG (H+L; #A11070) and Alexa Fluor 568 F(ab′) 2 fragment of goat anti‐mouse IgG (H+L; #A11019) were purchase from Life Technologies (Eugene, OR). A 1:500 dilution was for immunofluorescent staining.
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