Paraformaldehyde (pfa)

BrdU-labeled tissues were prepared for confocal microscopy by dissecting the thoracic ganglion in cold PBS using a dissecting microscope. The thoracic ganglion was stained with 25 nM MitoTracker Deep Red for 25 minutes (Invitrogen, Carlsbad, CA), rinsed with 0.1% Triton X-100 in PBS (wash buffer), and fixed for 30 minutes in 4% paraformaldehyde (Ted Pella Inc., Redding, CA) in PBS at 4°C. After rinsing in wash buffer, the thoracic ganglion was incubated in 1 M HCl for 30 minutes at 25°C, then blocked in 0.1% Triton X-100 with 10% heat-inactivated FBS in PBS (blocking buffer) for 1 hour at 25°C. Primary antibody to BrdU (Abcam #Ab6326, Cambridge, MA) was then added at 1∶100 dilution and incubated overnight at 4°C. After washing with wash buffer, Alexa Fluor 488 goat anti-rat IgG (Molecular Probes #A11006, Eugene, OR) was added to blocking buffer at 1∶10,000 dilution and incubated for 2 hours at 25°C. Stained thoracic ganglia were washed with wash buffer, mounted in Fluoromount (Sigma-Aldrich, St. Louis, MO), and imaged using an Olympus FV-1000 with a 100× lens and 2–4× digital zoom.

Rat hearts were perfused for 40 min with 4% paraformaldehyde and 2% glutaraldehyde (TedPella, Redding, CA, USA) in 0.1 mol/L phosphate buffer at a speed of 3 ml/min, after which the hearts were removed. Myocardial tissue was collected at one third above the apex cordis from the surrounding infarct region of left ventricle and cut into 1 mm³ blocks. The tissue blocks were fixed overnight at 4 °C with 3% glutaraldehyde, washed 3 times with 0.1 mol/L phosphate-buffered solution, and then post-fixed with 1% osmium tetraoxide for 2 h. The ultrathin sections were prepared as routine, observed and photographed with a transmission electron microscope (JEM 1230, JEOL, Tokyo, Japan)13 (link).

Mice were sacrificed >4–6 weeks after intravitreal injection of AAV2/2-hsyn-MW-coneopsin-YFP treatment. Eyes were isolated and fixed in 4% paraformaldehyde (Ted Pella) (30 min) and either flat mounted or embedded in agarose (Sigma) and sectioned in transverse using a vibratome (Leica Microsystems), as done previously^{19 (link),20 (link),22 (link)}. Retinal tissues were examined by confocal microscopy (Leica TCS SP5; Leica Microsystems). See Supplementary Methods for further description.

The normal breast tissue was obtained from a patient who was diagnosed with ductal cell carcinoma in situ (DCIS) at the age of 59 years and opted for single mastectomy of the afflicted breast. The patient was postmenopausal and nulliparous at the time of surgery. The pathology examination post surgery confirmed a 2 cm single-focus DCIS. Normal tissue was taken from a quadrant uninvolved in the tumor from the mastectomy specimen. The sample was collected and provided by the Biobank Core Facility of St. Joseph’s Hospital and Medical Center at the Barrow Neurological Institute according to the approved Institutional Review Board protocol #PHXA-05TS038. The normal tissue of ~3 g in weight was dissected and divided into several portions, one of which was fixed in 4% paraformaldehyde (Ted Pella, Inc., Redding, CA) in phosphate-buffered saline (PBS) at 4 °C overnight followed by paraffin embedding and H&E staining using a BOND-MAX autostainer (Leica Microsystems, Wetzlar, Germany). The other portion of ~1.5 g of tissue was rinsed in cold PBS and mechanically minced to 0.1 mm² in size using a scalpel. Tissue was frozen in 10% dimethyl sulfoxide (DMSO, Sigma, St. Louis, MO, USA) containing fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and stored at −80 °C.

All live and immunofluorescence microscopy was performed on a Nikon Eclipse Ti inverted fluorescence microscope. Image capturing was obtained using a Hamamatsu camera controller C10600 and Volocity imaging software, version 6.3 (PerkinElmer; Waltham, MA, USA). Infected cells were fixed in 3.7% paraformaldehyde (Ted Pella) for 15 m, then permeabilized with 0.1% Triton-X-100 (Fisher), blocked with 1% BSA-PBS (Fisher), and stained. Antibodies/dyes were obtained from the following sources: mouse anti-ALIX antibody from Santa Cruz biotechnology (Dallas, TX, USA), rabbit anti-VPS4 from Sigma-Aldrich (St. Louis, MO, USA), mouse anti-CEP55 from Santa Cruz Biotechnology, rabbit anti-CHMP4B from Santa Cruz Biotechnology, CellLight ER-RFP from Thermo Fisher (Waltham, MA, USA), Phalloidin 633, donkey anti-goat 488 from Invitrogen (Waltham, MA), DAPI, goat anti-mouse 488 from Thermo Fisher, anti-GFP 488, FM4-64 from Molecular Probes (Eugene, OR, USA), mouse anti-C. trachomatis LPS donated by Bob Suchland (University of Washington, WA, USA). Staining of abscission proteins: HeLa cells were seeded onto glass chamber slides and fixed with paraformaldehyde 24 h after seeding to stain for antibody localization of CHMP4B, VPS4, CEP55 and ALIX proteins. All abscission proteins are stained with antibodies labeled by GFP and nuclei is displayed by DAPI staining.