Anti mouse igg alexa fluor 594

Microencapsulated cells were collected from culture and fixed in 4% (wt/vol) formaldehyde with 4% (wt/vol) sucrose in phosphate-buffered saline (PBS) for 20 min at RT. For cryosectioning preparation, samples were dehydrated in 30% (wt/vol) sucrose overnight at 4°C, embedded in Tissue-Tek^® O.C.T. (Sakura) and frozen at -80°C. The frozen samples were sliced with a thickness of 10 μm in a cryostat (Cryostat CM 3050 S, Leica). The cryosections were permeabilized for 10 min with 0.1% (vol/vol) Triton X- 100 (Sigma–Aldrich) and blocked with 0.2% (wt/vol) fish-skin gelatin (FSG; Sigma–Aldrich) in PBS for 30 min. Primary and secondary antibodies were prepared in 0.125% (wt/vol) of FSG in PBS and incubated for 2 h. The primary antibodies used were as follows: cardiac troponin T (cTnT, Thermo Fisher Scientific, 1:200), sarcomeric α-actinin (Sigma–Aldrich, 1:200), collagen I (Abcam, 1:100), and collagen IV (Abcam, 1:100). Secondary antibodies used were as follows: anti–mouse IgG Alexa Fluor 594, anti–rabbit IgG Alexa Fluor 594, anti–mouse IgG Alexa Fluor 488, anti–rabbit IgG Alexa Fluor 488, and anti–mouse IgG Alexa Fluor 594 (all from Thermo Fisher Scientific, 1:500). The samples were mounted in Prolong^® Gold reagent containing DAPI (Life Technologies). Samples were visualized using a confocal fluorescence microscope (SP5, Leica).

Cells were plated and allowed to incubate on coverslips overnight. The following day the cells were fixed to coverslips using 4% Paraformaldehyde (PFA) Solution (Thermo Scientific). A permeablization step was not performed to preserve cell surface EGFR. After fixation, primary antibody EGFR (528) (Santa Cruz) antibody was used, followed by secondary antibody Alexa Fluor 594 anti-mouse IgG (Life Technologies). After antibody incubations, the coverslips were mounted to slides using ProLong Diamond Antifade Mountant with Dapi (Invitrogen). The slides were allowed to dry overnight (in the dark) at room temperature, and images were acquired with a Leica confocal microscope.

BMDMs were grown on 18-mm coverslips for 24 h at 37 °C. After Mtb infection for 3 h, the cells were fixed in 4% paraformaldehyde and washed three times with TBS-T. Next, the cells were blocked in 5% skim milk for 1 h at room temperature. The cells were cultured with primary antibodies overnight, and then with the appropriate secondary antibody (Alexa Fluor 594 anti-rabbit IgG, Alexa Fluor 594 anti-mouse IgG, Alexa Fluor 488 anti-rabbit IgG, and Alexa Fluor 488 anti-mouse IgG, Life Technologies, Carlsbad, CA, USA) for 2 h at room temperature. Next, the cells were stained with DAPI to label DNA. The stained cells were visualized under a DP70 fluorescence microscope (400× magnification; Zeiss, Oberkochen, Germany). Quantification of mitochondrial morphology was measured using ImageJ (NIH) as described previously [25 (link),26 (link)].