Retinal sections were deparaffinized with xylene and dehydrated in graded series of alcohols. Sodium citrate (10 mM, 0.05% Tween-20, pH 6.0) was used for the antigen retrieval for 20 minutes at boiling point. Sections were treated with 100 μL of rabbit anti active and procaspase 3 polyclonal primary antibody (ABCAM, Cambridge, MA, USA) diluted at 2:98 with 10 mM Tris 1% BSA and the incubation was performed overnight at 4°C. Subsequently, incubation with secondary antibody, which was conjugated with Texas red fluorochrome (goat anti rabbit IgG antibody - H&L Alexa Fluor® 594, ABCAM, Cambridge, MA), was done for 1 hour. Next, Tris-buffered saline (TBS) with 0.025% Triton X-100 was used to wash the sections for 5 minutes. The counterstaining was done for 10 minutes with 4,6-diamidino-2-phenylindole (DAPI) diluted in PBS 1 mM at a ratio of 1:999, and then the slides were mounted. The observations were made in a dark room using a fluorescence microscope (BX61TRF-FL-CCD; Olympus, Florida, USA). For Texas red 596 nm and for DAPI 358 nm filter was used.
H l alexa fluor 594
Manufactured by Abcam
Sourced in United States, United Kingdom
H&L Alexa Fluor® 594 is a fluorescent dye conjugate. It is designed to be used as a detection reagent in various immunoassays, flow cytometry, and microscopy applications.
Automatically generated - may contain errors
3 protocols using h l alexa fluor 594
1
Immunohistochemical Analysis of Apoptosis
2
Osteoblastic Differentiation Evaluation
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MLO-Y4 cells were detected on day 2. To evaluate osteoblastic differentiation, MC3T3-E1 cells were examined after seven days of osteogenic induction. Cells were incubated with a mixture of β-catenin (Proteintech, Rosemont, IL, USA, 66379) and Cx43 antibodies overnight and then incubated with a mixture of donkey anti-mouse IgG H&L (Alexa Fluor® 594) (Abcam, 150108) and goat anti-rabbit IgG H&L secondary antibodies. Finally, autofluorescence was quenched using the Auto Fluo Quencher C1212 kit. The cells were observed at excitation wavelengths of 488 and 594 nm.
3
Immunohistochemistry of Collagens I, II, and X
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5 µm thick paraffin-embedded pellets were deparaffinized, rehydrated, and then treated with protease XXV (AP-9006-005, Thermo Scientific) and hyaluronidase (H6254, Sigma-Aldrich). Sections were then incubated with primary antibody: rabbit anti-collagen I (CL50111AP-1, Cedarlane, ON, Canada), mouse anti-collagen II (II-II6B3, Developmental Studies Hybridoma Bank, IA, USA) using a 1:200 dilution and rabbit anti-collagen X (58632, Abcam, UK) using 1:100 dilution at 4 °C overnight, followed by incubation with a goat anti-rabbit IgG (H&L Alexa Fluor 594, Abcam, UK) with a 1:200 dilution for collagen I, X and goat anti-mouse IgG (H&L Alexa Fluor 488, Abcam) with a 1:200 dilution for collagen II. Sections were then stained with DAPI (4′, 6-diamidino-2-phenylindole, Cedarlane) and mounted with Glycerol and PBS (1:1 ratio). Immunofluorescence was visualized by an Eclipse Ti-S microscope (Nikon Canada, Mississauga, Canada).
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