Fluorescence Microscopy

All reagents were obtained from Sigma Chemical Co. (St. Louis, MO, USA) unless otherwise indicated. HeLa cells were grown for 1-2 days in Dulbecco's modified Eagle's medium (HyClone, Logan, VT, USA) and 10% (v/v) calf serum (Intergen Co., Purchase, NY, USA) on 19 mm cover slips coated with 0.1% (w/v) type I collagen in 0.1 M acetic acid. They were then fixed for 10 min with 2% paraformaldehyde in phosphate-buffered saline (PBS: 140 mM NaCl, 2.6 mM KCl, 8.1 mM Na₂HPO₄, 1.5 mM KH₂PO₄, 0.9 mM CaCl₂, 0.5 mM MgCl₂, pH 7.4), permeabilized for 10 min with 0.1% saponin in cytoskeletal stabilization buffer (CSB: 137 mM NaCl, 5 mM KCl, 1.1 mM Na₂HPO₄, 0.4 mM KH₂PO₄, 4 mM NaHCO₃, 2 mM MgCl₂, 2 mM EGTA, 5 mM Pipes, 0.1% glucose, pH 6.1) and incubated for 60 min with a primary antibody.

Monoclonal antibodies directed against an ER antigen (DAKO, Carpinteria, CA, USA), the Golgi protein giantin [23], the Golgi protein GPP130 [43], the lysosomal protein LAMP2 [44], a mitochondrial outer membrane protein (Serotec, Oxford, England), the nucleolar protein nucleolin [45], transferrin receptor (O.E.M. Concepts, Toms River, NJ, USA), and tubulin (Sigma) were used as primary antibodies in separate labeling experiments. Working dilutions of antibody stock solutions were obtained by empirically optimizing for low background in the presence of adequate specific signal (see Table 3.1). Filamentous actin was labeled with 53 nM rhodamine phalloidin (Molecular Probes).

 

 

Table 3.1: Concentration and clone number (if applicable) for the ten labels used with the HeLa cells.

Label	Working Concentration	Clone
anti-ER protein	Stock diluted 1:25	RFD6
anti-giantin	Supernatant concentrated	133
	$\sim$10X with ammonium sulfide,
	then diluted 1:50
anti-gpp130	Supernatant concentrated
	$\sim$10X with ammonium sulfide,
	then diluted 1:50
anti-LAMP2	1:1 (supernatant)	H4B4
anti-mitochondrial protein	50 $\mu$g/ml	MOM/H6/C12
anti-nucleolin (Cy3)	20 $\mu$g/ml
rhod. phalloidin	53 nM
anti-transferrin receptor	40-140 $\mu$g/ml	236-15375
anti-tubulin	7 $\mu$g/ml	2-28-33
DAPI	10 $\mu$g/ml
anti-mouse IgG (Cy5)	12.5 $\mu$g/ml

After 3 washes of 5 min each in CSB, the samples requiring a secondary antibody were incubated for 45 min with 12.5 $\mu$g/ml of a Cy5-conjugated anti-mouse IgG antibody (Jackson Immunoresearch, West Grove, PA, USA). All samples were incubated with 10 $\mu$g/ml DAPI (Molecular Probes, Inc., Eugene, OR USA) to label DNA. The coverslips were washed three more times in CSB before mounting on microscope slides using gelvatol (60 ml of 10 mM Tris, 15 g Airvol 205 (Air Products, Allentown, PA, USA), 30 ml glycerol, 1 g n-propyl gallate). Images of Cy5 and DAPI fluorescence were acquired separately using a Zeiss Plan-Neofluar objective (100x, 1.3 NA), and a Photometrics CH 250 cooled charge-coupled device (512 x 382 23 $\mu$m square pixels) mounted on a customized Zeiss Axiovert microscope [22].

Each slide was scanned for single cells that were spread out on the coverslip (i.e., not rounded up in mitosis). Each such field of view was acquired as a stack of three images in which the sample was moved up, through focus 0.237 $\mu$m between each slice. The output of the microscopy steps for each selected field of view consisted of two stacks of images, one depicting the protein localization pattern and the other showing the localization of DNA.