Super coolscan 9000

To perform the cryo-EM experiments, 3.4 μl of sample were deposited in a c-flat grids (CF-2/2-2C) with an additional continuous layer of thin carbon (5–10 nm). Grids were glow discharged in air at 5 mA for 15 s before sample addition. Samples contained the Dox–PoP-liposomes before and after irradiation at a concentration of 5 mg ml⁻¹ (Dox concentration ~1.5 mg ml⁻¹) in a buffer with 10 mM HEPES (pH 7.4) and 10% sucrose. Grids were blotted twice and vitrified by rapidly plunging them into liquid ethane at −180 °C using a Vitrobot (FEI). The blotting chamber of the Vitrobot was set up at 25 °C and 100% relative humidity. Grids were transferred to a JEOL 2010 F electron microscope operated at 200 kV using a Gatan 914 cryo-holder. Images were recorded on Kodak SO-163 films under low dose conditions (~15–20 e per Ų) at a nominal magnification of × 50,000 and a defocus of −5 μm. Electron micrographs were digitized with a step size of 12.7 μm in a Nikon Super Coolscan 9000 scanner producing images with a sampling value of 2.54 Å per pixel.

Kidneys and ventricle tissue fixed in formalin were sent to the Children’s Research Institute Histology Core at the Medical College of Wisconsin, where they were processed, paraffin embedded, sectioned (4 µM), mounted on slides, and Masson’s trichrome stained. Trichrome stained kidney sections were then imaged at 20 × using an E-400 microscope (Nikon) and SPOT Insight V5.1 digital camera (Diagnostic Instruments). Ventricle sections were scanned using a Super Coolscan 9000 (Nikon). The area of extracellular matrix protein containing (i.e. fibrotic; blue) tissue was quantified using Metamorph software and expressed as fibrosis percentage of total tissue area^{40 (link)}.
Immunohistochemistry was performed for detection of CD177 antigen, as previously described^{38 (link)}. CD177 antibody (sc-376329; Santa Cruz Biotechnology) was used at a 1:50 dilution and a rat adsorbed anti-mouse secondary antibody (#BA-2001; Vector Laboratories) was used at a 1:200 dilution. A secondary control slide (no primary antibody; 1:200 secondary antibody) slide was run in parallel with no staining observed.

Samples of HIV-1 gp120-14K were applied onto carbon-coated copper grids and stained with 2% uranyl acetate. Micrographs were taken under minimal dose conditions in a JEOL JEM1200EXII microscope operated at 100 kV and digitized in a Nikon Super CoolScan 9000 scanner with a pixel size of 2.12 Å/pixel. Individual particles were manually selected using XMIPP3.1 [40 (link)]. Image classification was performed using multi-reference free pattern based on correlation and standard maximum correlation criterion refinement (CL2D), as implemented in XMIPP3.1 [41 (link)]. Homogeneous populations were obtained and averaged for a final two-dimensional characterization.

The heart was dissected and fixed in 4% paraformaldehyde overnight at 4 °C. The tissues were then washed with PBS and embedded using sucrose and OCT. Frozen hearts were sectioned onto slides in 8 micron thick axial sections. Slides were stained with Masson’s Trichrome stain and hematoxylin counterstain (Masson Trichrome Special Stain Kit, Leica, Buffalo Grove, Illinois). Images of the entire section were obtained (Nikon Super Coolscan 9000). Metamorph image analysis software was used to quantify percent fibrosis by means of thresholding the specific blue coloration of collagen positive regions. The thresholded area was compared to total area of cardiac tissue and represented as percentage of total tissue area. An average of two sections for each sample was used for each measurement.

Upon sacrifice, tumors were collected and fixed in formalin and paraffin embedded. Immunohistochemistry staining was performed on 5 μM sections using standard streptavidin-biotin-peroxidase procedure with a Ventana NexEs autostainer and the solvent-resistant DAB Map Detection Kit. Antibodies (p27KIP, 1:100; Ki-67, ready to use) were purchased from Dako Canada or (PARP Asp214, 1:200) from Cell Signaling. Proliferation index was determined by the nucleus count (Ki-67⁺ cells/total cells) in sampled fields, then normalized relative to control tumor proliferation index. An average of 3–4 fields per tumor were sampled (n = 12 control tumors and 14 treated tumors). Immunostaining quantitation of P27^KIP was performed using a SuperCoolScan9000 (Nikon, USA) and Yellow channel was extracted from a CMYK color model using Fiji Software (Open Source) [47 (link)]. To avoid any off-tumor quantitation, tumors were counterstained with standard hematoxylin and eosin procedures. Apoptosis index was determined using the same method as Ki-67 counts. Pictures with 100 × and 400 × magnifications were acquired using an Axioskop2 phase-contrast microscope (Carl Zeiss, USA) and processed using ImagePro software (Media Cybernetics, USA).