Eyes were enucleated, and retinae were dissected and fixed in 4% formaldehyde for 30 min at room temperature. Fixed retinae were cryoprotected in sucrose in PBS for a few hours and embedded in optical cutting temperature compound on dry ice. Sections (20 μm thick) were cut on a cryostat (Leica). Retinal sections or whole retinal cups were blocked in 5% BSA in PBST (PBS with 0.1% Triton X-100), stained with primary antibodies at 4 °C overnight, and washed three times with PBST. Primary antibodies used in this study included rabbit anti-red/green opsin (1:300, AB5405; EMD Millipore); goat anti-blue opsin (1:100, sc-14365; Santa Cruz Biotechnology Inc.); rabbit anti-GFAP (1:500, Z0344; DAKO); rabbit anti-Iba1 (1:1,000, PA5-21274; ThermoFisher), and rhodamine-conjugated and FITC-conjugated PNA (1:1,000; Vector Laboratories). Sections were stained using secondary antibodies, including donkey anti- rabbit CY3, donkey anti-rabbit Alexa Fluor 647, and donkey anti-goat Alexa Fluor 647 (all used at 1:1,000; Jackson ImmunoResearch), and were costained with DAPI in the dark for 2 h at room temperature and mounted in Fluoromount-G (SouthernBiotech). Images were taken using a 40× objective with Z-stacks on a Zeiss LSM780 confocal microscope. Images used for comparison between groups were taken side by side at the same confocal settings.
Z0344
Manufactured by Agilent Technologies
The Z0344 is a high-precision laboratory instrument designed for accurate measurements and analysis. It features advanced technology to ensure reliable and consistent performance. The core function of this product is to provide users with precise data and insights, enabling them to make informed decisions in their research or industrial applications.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Sc 14365, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti iba1, by Thermo Fisher Scientific (1 mentions)
Donkey anti rabbit alexa fluor 647, by Jackson ImmunoResearch (1 mentions)
Donkey anti goat alexa fluor 647, by Jackson ImmunoResearch (1 mentions)
Donkey anti rabbit cy3, by Jackson ImmunoResearch (1 mentions)
Cryostat, by Leica camera (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
Ab5405, by Merck Group (1 mentions)
Species specific hrp conjugated secondary antibodies, by Jackson ImmunoResearch (1 mentions)
Mab4423, by Merck Group (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Bradford reagent, by Bio-Rad (1 mentions)
2 protocols using z0344
1
Immunohistochemical Analysis of Retinal Cell Types
2
Protein Lysate Preparation and Western Blot Analysis
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Cells were lysed in M2 Buffer (20 mM Tris HCl, pH 7.6, 0.5% NP40, 250 mM NaCl, 3 mM EDTA and 3 mM EGTA), supplemented with 1×EDTA-free protease inhibitor cocktail (MedChemExpress, HY-K0010). Protein concentration was quantified by the Bradford reagent (BIO-RAD, 500-0205). Protein samples were mixed with Laemmli loading buffer and boiled for 10 minutes. Subsequently, samples were separated by SDS polyacrylamide gelelectrophoresis (SDS-PAGE), then were transferred to polyvinylidene fluoride (PVDF) membranes (PALL, BSP0161). Blots were incubated with primary antibodies overnight at 4°C followed by HRP-conjugated species-specific secondary antibodies (Jackson Immuno Research, 1:5000) at room temperature for one hour. Finally, the signals were detected by ECL luminescence reagent (Absin, abs920). The following antibodies were used at the indicated dilutions: WDR12 (1:1000, NBP1-53111, Novus), Pes1 (1:1000, b88543, Abcam), Bop1 (1:1000, 28366-1-AP, Proteintech), SOX2 (1:1000, MAB4423, Millipore), Olig2 (1:1000, sc-48817, Santa Cruz), GFAP (1:1000, Z0344, Dako), GAPDH (1:1000, 3683s, Cell Signaling).
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