The immunostaining assay protocol was modified from the study by Wagner and Levine (58 (link)). In brief, the larvae were fixed at 20 or 24 hours after fertilization in MEM-FA. After washes in PBT, pretreatment in PBS with 0.1% Triton, washes in PBT, and incubation for 30 min in blocking solution at room temperature [10% Western blocking solution (Sigma-Aldrich) in PBT], the larvae were incubated at 4°C overnight with anti–acetylated tubulin antibody (1:500; Sigma-Aldrich, T7451) in blocking solution. After washes in PBT, the primary antibody was detected using goat anti-mouse antibody conjugated to Alexa Fluor 555 (1:1000; Thermo Fisher Scientific, A21424) diluted in blocking solution for 45 min at room temperature. The larvae were washed several times in PBT and mounted on slides using FluorSave Reagent (Millipore).
Western blocking solution
Manufactured by Merck Group
Western blocking solution is a laboratory reagent used in Western blot analysis. It functions to block nonspecific binding sites on a membrane, preventing the attachment of antibodies to areas other than the target proteins. This helps to improve the specificity and signal-to-noise ratio of the Western blot assay.
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Lab products found in correlation
2 protocols using western blocking solution
1
Immunostaining Assay for Acetylated Tubulin
2
Protein Extraction from Worm Samples
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To generate protein lysates, animals were washed in M9, then in sterile water. Pelleted animals were resuspended in equal volumes of worm lysis buffer (Webster et al., 2013 (link)), incubated at 85°C for 5 min, then sonicated on ice for two cycles of 30 s each at 70% power using a Sonic Dismembrator Model 100 (Fisher Scientific). Homogenates were quantified using a DC Protein Quantification Kit (BioRad). Protein (50 μg per lane) was loaded using Criterion systems (BioRad) and transferred to Immobilon Psq (Millipore). Details pertaining to antibodies are provided in the supplementary Materials and Methods . Membranes were blocked with 5% milk in TBS+0.5% Tween 20. Antibodies were diluted in Western Blocking Solution (Sigma) prior to incubation with membranes. Blots were washed with TBS+0.5% Tween 20. Signal was detected by ECL (Pierce).
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