monoclonal antibody to LC3

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	- Immunohistochemistry (IHC) Ab is incubate for overnight at 4C.

Protocol tips
- Immunohistochemistry (IHC) Ab is incubate for overnight at 4C.

Publication protocol

Immunohistochemistry (IHC)
Paraffin-embedded intestinal sections were deparaffinized at 56°C, immersed in xylene three times and hydrated with ethanol (two times with 100%, two times with 95% and one time with 75% ethanol) for 5 minutes each. For antigen unmasking, slides were heated in 10 mM sodium citrate buffer (pH 6.0) for 10 minutes prior to treatment with 3% hydrogen peroxide for 10 minutes. The specimens were treated with 5% BSA in TBST (Tris-buffered saline with 0.1% v/v Tween-20) for 1 hour at room temperature followed by overnight incubation with a rabbit polyclonal Beclin 1 antibody (ProSci, Inc., Poway, CA), rabbit polyclonal cleaved caspase-3 antibody (Cell signaling, Danvers, MA) or mouse monoclonal anti-LC3 antibody (nanoTools, Teningen, Germany) at 4°C. After washing, the sections were incubated with anti-rabbit HRP or anti-mouse HRP for 30 minutes at room temperature (Dako, Carpinteria, CA). Positive staining was visualized with DAB chromogen and nuclei counterstain was performed with hematoxylin. Images were acquired by Olympus FSX100. For quantitation of Beclin 1, LC3, and cleaved caspase-3 staining, the intensity of IHC staining was scored on a “0” to “3” scale with “0” being no staining, “1” being weak staining, “2” being moderate staining and “3” being strong staining among slides examined [40].

Immunofluorescence (IF)
After deparaffinization and rehydration, intestinal sections were blocked in 5% normal goat serum (Sigma, St. Louis, MO) for 1 hour at room temperature and then incubated with mouse monoclonal LC3 antibody (nanoTools, Teningen, Germany) and mouse monoclonal p62 antibody (BD Biosciences, San Jose, CA) at 1∶100 dilution, followed with an Alexa Fluor 488-conjugated second antibody (Molecular Probes, Eugene, OR). Nuclei were labeled with 4′, 6-diamidino-2-phenylindole (DAPI) (100 ng/ml) (Sigma, St. Louis, MO). Coverslips were mounted on slides using SlowFade Gold anti-fade reagent (Invitrogen, Grand Island, NY). Images were acquired by Olympus DSU Spinning Disk Confocal and analyzed using Slidebook Software (Olympus Inc., Center Valley, VA).

Immunoblotting
For tissue lysate preparation, small intestinal tissues were minced and sonicated in 300 µl RIPA buffer (50 mM Tris•HCl, pH 7.4; 150 mM NaCl; 1% Nonidet P-40 (NP-40); 0.1% sodium dodecyl sulphate (SDS); 0.5% sodium deoxycholate) containing protease inhibitors (Roche, Indianapolis, IN) and phosphatase inhibitor (Thermo Scientific, Rockford, IL). For cell culture lysate preparation, cells on plates were rinsed with cold PBS and lysed in the same lysis buffer used for tissue lysate preparation. The total protein concentration in lysates was determined using a BCA™ protein assay kit (Pierce, Rockford, IL). Lysates were resolved on SDS–PAGE. After electrophoresis, proteins were blotted onto a nitrocellulose membrane. The blots were blocked in 5% milk PBS-T (0.1% Tween 20 (v/v) in phosphate-buffered saline) for 1 hour at room temperature and then probed with primary and appropriate secondary antibodies in 5% milk PBS-T. Immunoreactive bands were visualized by chemiluminescence reaction using ECL reagents (Pierce, Rockford, IL) followed by exposure of the membranes to autoradiography film (Denville scientific, Metuchen, NJ). Protein levels were quantified by densitometry using ImageJ software. The LC3 antibody was purchased from nanoTools (Teningen, Germany), the Beclin 1 antibody was from ProSci (Poway, CA), the p62 antibody was from BD Biosciences (San Jose, CA), the phospho-Akt, phospho-mTOR, phospho-p70S6K, phospho-p44/p42 MAPK, Akt, phospho-Jak2, Jak2 and Bcl-2 antibodies were purchased from Cell signaling (Danvers, MA). The β-actin antibody was from Sigma (St. Louis, MO). Protein bands of interest were quantitated by densitometry and normalized to β-actin.

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