Alexa fluor 488 conjugated f ab fragment anti rabbit igg

Manufactured by Immunological Sciences

Alexa-Fluor-488-conjugated F(ab′) fragment anti-rabbit IgG is a laboratory reagent used for immunofluorescence applications. It consists of an F(ab') fragment of an antibody that specifically binds to rabbit immunoglobulin G (IgG), conjugated with the Alexa Fluor 488 fluorescent dye.

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Lab products found in correlation

Psmad3 primary antibody, by Abcam (2 mentions) Tcs sp8, by Leica (2 mentions) Sp5 confocal microscope, by Leica (1 mentions) Sterile glass coverslips, by Marienfeld (1 mentions) Lc3 primary antibody, by Cell Signaling Technology (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)

3 protocols using alexa fluor 488 conjugated f ab fragment anti rabbit igg

Quantifying Autophagy Induction in Fibroblasts

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A total of 1.5×10⁴ fibroblasts were plated on sterile glass coverslips (Marienfeld) in 24-well plates in triplicate. After 5 days, cells were treated for 6 h with 10 μM chloroquine. Following the treatment, the medium was removed and cells were fixed with cold 100% CH₃OH for 15 min at −20°C, washed three times with PBS and blocked for 1 h in 1% BSA in PBS containing 0.3% Triton X-100. Then, cells were incubated with LC3 primary antibody (Cell Signaling) diluted 1:500 in 1% BSA, 0.3% Triton X-100 in PBS overnight at 4°C. Cells were washed three times with PBS and incubated with secondary antibody [Alexa-Fluor-488-conjugated F(ab′) fragment anti-rabbit IgG, Immunological Sciences] diluted 1:2000 in 1% BSA, 0.3% Triton X-100 in PBS for 2 h at RT. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). The samples were analyzed using an SP5-Leica confocal microscope (Leica). The total area of punctate signal per cell was measured by the Leica software LAS4.5.

Besio R., Garibaldi N., Leoni L., Cipolla L., Sabbioneda S., Biggiogera M., Mottes M., Aglan M., Otaify G.A., Temtamy S.A., Rossi A, & Forlino A. (2019). Cellular stress due to impairment of collagen prolyl hydroxylation complex is rescued by the chaperone 4-phenylbutyrate. Disease Models & Mechanisms, 12(6), dmm038521.

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Quantifying pSMAD3 Signaling in TGF-β Treated Cells

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2×10 4 OBs were plated on glass coverslips in 24 well plate. The next day cells were treated for 24 h with 1 ng/mL TGF-β. Cells were fixed with 10% neutral buffered formalin for 30 min at RTand blocked 1 h in 1% BSA in PBS containing 0.3% TritonX100. pSMAD3 primary antibody (Abcam) was used at 1:500 dilution in 1% BSA, 0.3% TritonX100 in PBS and the incubation was carried out o/n at 4°C. Cells were incubated with secondary antibody (AlexaFluor 488 conjugated F(ab') fragment anti-rabbit IgG, Immunological Sciences) 1:2000 in 1% BSA, 0.3% TritonX100 in PBS for 2 h at RT. Nuclei were stained with DAPI and images were acquired by confocal microscope TCS SP8 (Leica). The total area of punctate signal per cell and of punctate signal per nucleus were measured by the Leica software LAS 4.5.

Besio R., Contento B.M., Garibaldi N., Filibian M., Sonntag S., Shmerling D., Tonelli F., Biggiogera M., Brini M., Salmaso A., Jovanovic M., Marini J.C., Rossi A, & Forlino A. (2023). CaMKII inhibition due to TRIC-B loss-of-function dysregulates SMAD signaling in osteogenesis imperfecta. Matrix biology : journal of the International Society for Matrix Biology, 120.

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Quantifying TGF-β-Induced pSMAD3 Punctate Signaling

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2×10⁴ OBs were plated on glass coverslips in 24 well plate. The next day cells were treated for 24 h with 1 ng/mL TGF-β. Cells were fixed with 10% neutral buffered formalin for 30 min at RTand blocked 1 h in 1% BSA in PBS containing 0.3% TritonX100. pSMAD3 primary antibody (Abcam) was used at 1:500 dilution in 1% BSA, 0.3% TritonX100 in PBS and the incubation was carried out o/n at 4°C. Cells were incubated with secondary antibody (AlexaFluor 488 conjugated F(ab’) fragment anti-rabbit IgG, Immunological Sciences) 1:2000 in 1% BSA, 0.3% TritonX100 in PBS for 2 h at RT. Nuclei were stained with DAPI and images were acquired by confocal microscope TCS SP8 (Leica). The total area of punctate signal per cell and of punctate signal per nucleus were measured by the Leica software LAS 4.5.

Joiner W.J., Wang L.Y., Tang M.D, & Kaczmarek L.K. (1997). hSK4, a member of a novel subfamily of calcium-activated potassium channels. Proceedings of the National Academy of Sciences of the United States of America, 94(20), 11013-11018.

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