Adult P90 mice were anaesthetized and perfused through the heart with 30 ml PBS, followed by first 150 ml of a 5% glutaraldehyde/4% PFA/0.1 mM phosphate buffer fixative solution, then a 4% PFA/0.1 mM phosphate buffer fixative solution. Brains were dissected and post-fixed in 4% PFA/PBS for 30 minutes at 4°C. Coronal vibratome free-floating sections of 50 μm were collected and immersed in 0.1% sodium borohydride/PBS for 20 minutes at room temperature, blocked with 10% goat serum/0.5% gelatin/PBST (0.01% Triton X-100), and incubated with α-Cav-1 (1:100, Cell Signaling Technologies #3267; RRID:AB_2275453) overnight at room temperature. Sections were then incubated with gold-labeled goat α-rabbit IgG (1:50, Nanoprobes) overnight at room temperature, washed with PBS and sodium acetate, and silver-enhanced prior to processing for TEM.
α cav 1
Manufactured by Cell Signaling Technology
α-Cav-1 is a polyclonal antibody that recognizes the α-subunit of the caveolin-1 protein. Caveolin-1 is a structural component of caveolae, which are invaginations of the plasma membrane involved in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Lc3a b 1 2, by Cell Signaling Technology (1 mentions)
Pink1, by Cell Signaling Technology (1 mentions)
Parkin, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
Bradford reagent, by Bio-Rad (1 mentions)
Sds page, by Bio-Rad (1 mentions)
2 protocols using α cav 1
1
Ultrastructural Localization of Caveolin-1
2
Western Blot Analysis of Cav1, Autophagy, and Mitophagy
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Cells were washed three times with ice-cold 1× PBS (Gibco), and then lysed with RIPA Buffer including Protease and Phosphatase Inhibitors (Roche Lifesciences). Protein concentration was measured using the Bradford reagent (BioRad) and equal amounts of proteins (20 µg per lane) were resolved on SDS-PAGE (BioRad). Proteins were electro-transferred onto polyvinylidene-difluoride (PVDF) membrane. After blocking with 5% nonfat milk, blots were incubated with α-Cav1 (Cell Signaling, Cat# 3267S, 1:1000 dilution), GAPDH (Cell Signaling, Cat# 2118, 1:1000 dilution); autophagy marker LC3A/B-I/II (Cell Signaling, Cat# 12741, 1:1000 dilution) or mitophagy markers Parkin and PINK1 (Cell Signaling, Cat# 4211 and 6946, respectively, both at 1:1000 dilution). Uncropped original immunoblot scans are provided in Supplementary Fig. 9 .
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