Anti-LC3B antibody produced in rabbit

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	primary antibody dilution 1:750

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primary antibody dilution 1:750

Publication protocol

Immunostaining
Paraffin wax was removed from mouse lung tissue or hTEC ALI paraffin-embedded sections by incubation with xylene, followed by hydration in three rinses of isopropyl alcohol for 5 min each. Antigen retrieval was accomplished by boiling tissues in Trilogy solution (Cell Marque; 920P-09) for 20 min. Non-specific binding was blocked in 5% donkey serum in 0.1% Triton ×100. Lectin-UEA1 (Vector Laboratories; #RL 1062) was used to identify mucous cells in mouse lung sections. The mouse monoclonal antibody (Thermo Scientific; #MA1-38223) against MUC5AC 45M1 epitope was used in hTEC sections to mark MUC5AC staining. The rabbit polyclonal LC3B (microtubule-associated proteins 1A/1B light chain 3B) antibody (Sigma-Aldrich; #L7543) was used to immunostain autophagosomes in both mouse lung sections and hTEC ALI sections. A mouse monoclonal LC3B antibody (Enzo ALX; 803-080-C100) was used to immunostain LC3B in hTEC when used in combination with anti-DUOX1. A rabbit polyclonal antibody was used for DUOX1 [28] immunostaining. Fluorophore-labeled donkey secondary, species-specific antibodies were Alexa Fluor 488 or 555 (Life Technologies; #A-31570, #A21202, #A21206, #A31572). To localize DUOX1 on hTEC cultured on glass cover slips, fixed cells were blocked with 5% donkey serum in 0.1% Triton ×100 in PBS for 1 h at room temperature. Anti-DUOX1 antibody (1:500) was incubated with cells overnight at 4 °C. After three PBS washes, cells were incubated with fluorescent labeled donkey anti-rabbit secondary antibody and phalloidin (Thermo Fischer; #A12379) conjugated to Alexa 488 nm fluorophore to identify filamentous actin. Cells were incubated for 30 min at room temperature and then washed again with PBS.


LC3B flux assay
In vitro autophagy activity was determined by the flux assay for LC3B using chloroquine (50 μM) for transient autophagosome-lysosome disruption as previously described [21], [44]. LC3B II and I forms were distinguished by relative molecular weight after separating proteins on a 15% agarose gel. Cells were washed with cold PBS and lysed using RIPA buffer (Sigma-Aldrich; #R0278) containing protease inhibitors (Sigma-Aldrich; #P8340). Protein-containing supernatants were isolated by centrifugation, denatured at 100 °C for 5 min, then briefly disrupted using a water bath sonicator (Branson 5510 model) in four 30-second bursts, and mixed with Laemmli buffer containing 2-mercaptoethanol. Autophagy marker LC3B II levels were assayed by immunoblot using LC3B antibody (1:750 dilution; Sigma-Aldrich; #L7543) on 0.45 µm polyvinylidene fluoride (PVDF) membranes (Immobolin FL; Sigma-Aldrich; IPFL00010). Horseradish peroxidase conjugated goat anti- mouse or rabbit antibodies, respectively, were then used to image primary antibodies with densitometry values normalized to actin. To determine the relevance of STAT6 signaling in autophagy induction, hTEC were lysed in buffer containing NP-40 detergent (0.5%) with 50 mM HEPES (pH 7.0), 5 mM EDTA, 50 mM NaCl, 10 mM Na Pyrophosphate, 50 mM Na fluoride, protease inhibitors (Roche, #11697498001), 100 μM sodium orthovanadate, and freshly prepared phenylmethanesulfonylfluoride (1 mM) dissolved in 200 proof isopropanol. Lysates were centrifuged at 9800g 4 °C. Supernatants were then collected and run on 6–15% gradient acrylamide gel and transferred on PVDF membranes. Proteins were blocked in 5% bovine serum albumin for 1 h and then incubated with primary antibodies to STAT6 (1:500 dilution; Cell Signaling; #D3H4) or phosphorylated STAT6 (Tyr641; 1:500 dilution; Cell Signaling; #C11A12) overnight at 4 °C. STAT6 proteins were detected by chemiluminescence as described above.

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