- Goat anti-guinea pig IgG (HþL) HRP-conjugated (Santa
Cruz, sc2438): for IB 1:5000 in TBS-T, 5% (w/v) skim milk
powder. - Rat anti-mouse IgG (H þ L) HRP-conjugated (Jackson
ImmunoResearch, 415-035-166): for IB 1:10,000 in TBS-T,
5% (w/v) skim milk powder. - Goat anti-rabbit IgG (H þ L) HRP-conjugated (Jackson
ImmunoResearch, 111-035-144): for IB 1:10,000 in TBS-T,
5% (w/v) skim milk powder. - Goat anti-mouse IgG (HþL) Alexa Fluor 488 (Invitrogen/
Thermo Fisher Scientific, A-11001): for IF 1:1000 in PBS, 1%
(v/v) goat serum.
2.6 Indirect
Immunofluorescence
and Confocal Laser-
Scanning Microscopy
- Tissue culture dishes: 12-well.
- Parafilm.
- Fixation solution: 3.7% (w/v) paraformaldehyde (PFA) in PBS.
Store at 20 C. - Permeabilization solution: 40μg/ml digitonin in PBS. Store at
4 C(seeNotes 7and 8 ). - IF blocking solution: 3% (v/v) goat serum in PBS.
- IF antibody dilution: 1% (v/v) goat serum in PBS.
- DNA staining solution (Hoechst): 1 mg/ml Hoechst 33258 in
H 2 O. Store at 4C protected from light (seeNote 9). - Mounting solution: 10% (w/v) MOWIOL 4-88, 0.1 M
Tris–HCl, pH 8.5, 25% (v/v) glycerol. Store long term at
20 C and short term at 4C(seeNotes 9and 10 ). - Microscope slides 7626 mm with frosted ends.
- Confocal laser-scanning microscope and instrument software.
- Precision tissue wipes.
- Ultrapure water.
- ImageJ software (National Institute of Health, Bethesda).
3 Methods
If not stated otherwise, carry out all procedures at room tempera-
ture and according to the manufacturer’s instructions.3.1 Cultivation
of Cell Lines
and Stimulation
- Culture U2OS and NIH3T3 cells in DMEM high glucose at
37 C in an incubator providing saturated humidity and 5%
CO 2. Add 1μg/ml puromycin to the medium of NIH3T3 cells
stably expressing the double-tagged mCherry-EGFP-LC3
construct. Generally, grow the cells in 10 cm dishes and passage
every 2–4 days (seeNotes 11and 12 ).
378 Sarah Krieg et al.