For stable signal generation in imaging and flow cytometry, Alexa Fluor 488 dye is pH-insensitive over a wide molar range. Invitrogen™ Alexa Fluor 488 dye is a bright, green-fluorescent dye with excitation ideally suited to the 488 nm laser line. The benefits of this extra step are apparent in multiplexing/multicolor-staining experiments (e.g., flow cytometry) where there is potential cross-reactivity with other primary antibodies or in tissue/cell fluorescent staining experiments where there are may be the presence of endogenous immunoglobulins.Īlexa Fluor dyes are among the most trusted fluorescent dyes available today. Only the nonspecific-binding secondary antibodies are captured in the column, and the highly specific secondaries flow through. The secondary antibody solution is passed through a column matrix containing immobilized serum proteins from potentially cross-reactive species. Cross-adsorption or pre-adsorption is a purification step to increase specificity of the antibody resulting in higher sensitivity and less background staining. To minimize cross-reactivity, these chicken anti-goat IgG (H+L) whole secondary antibodies have been affinity purified and cross-adsorbed against human, mouse, and rabbit IgG prior to conjugation. j, k Quantification of the ciliary length (j) and ARP-T1 protein … Image collected and cropped by CiteAb from the following publication (), licensed under a CC BY license. Cell nuclei are stained with DAPI (blue). i Immunofluorescence stainings of acetylated-tubulin (green) and rootletin (pink) in 48 h serum-starved control and ACTRT1 KD hTERT-RPE1 cells. h Quantification of ciliary length of (g). g Immunofluorescence stainings of acetylated-tubulin (green) and rootletin (pink) in 48 h serum starved hTERT-RPE1 cells expressing an empty vector (V), or ACTRT1 mutant (M), or ACTRT1 WT (WT). e, f Correlation between the ARP-T1 fluorescence and ciliary length (e), and between the ARP-T1 and rootletin fluorescence (f). c, d Quantification of the relative fluorescence intensity of rootletin (cN = 5) and ARP-T1 (dN = 10) on the ciliary rootlet. b Quantification of the ciliary length from 3D confocal immunofluorescence microscopy images. The Bazex−Duprv©−Christol syndrome is a ciliopathy caused by ARP-T1 loss of function, and knockdown of ACTRT1 in hTERT-RPE1 cells induces resorption of primary cilia.a Representative immunofluorescence images using acetylated-tubulin (green) and rootletin (red) (top), and ARP-T1 (green) and rootletin (red) (bottom) in hair follicle, sporadic BCC and 4 BDCS (ACTRT1 547_548insA, mutation B2, mutation A3, mutation CNE126). The images were captured at 40X magnification. Nonspecific staining was not observed with secondary antibody alone (panel f). The specificity of the secondary antibody was proved by the absence of signal in HeLa(negative model for CD10) due to no primary antibody binding (Panel e). F-actin was stained Alexa Fluor™ 647 Phalloidin (Product # A22287, 1:1000) (Panel c: red). Nuclei (Panel b: blue) were stained with Hoechst33342 (Product # H1399). Chicken anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488 (Product # A-21467, 1:2000 dilution) in 0.1% BSA in PBS for 45 minutes at room temperature was used for detection of CD10 on the plasma membrane (Panel a: Green). The cells were fixed with 4% paraformaldehyde for 10 minutes, blocked with 2% BSA for 1 hour and labeled with 1:100 dilution of primary antibody overnight at 4 degrees celsius. Immunofluorescence analysis of Chicken anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488 (Product # A-21467) was performed using LNCaP (positive model) and HeLa (negative model) cells stained with CD10 Polyclonal Antibody (Product # PA5-47075).
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