Axiovert 35 microscope

Mouse aortas were perfused with PBS followed by 10% formalin or 4% paraformaldehyde. Aortic root sections were embedded in OCT for frozen sectioning. Photos of aortic root sections were taken with an Axiovert 35 Zeiss microscope (Zeiss, Germany) equipped with an Axiocam CCl camera at × 25 magnification. En face stained aortas and light microscopy was obtained with a Nikon Stereo microscope equipped with a Spot Insight camera (Spot Imaging) at × 5 or × 10 magnification. Aortic root sections and en face preparations were stained with Oil Red-O in 60% isopropanol or 80% methanol. Aortic roots were additionally stained with haematoxylin and eosin or trichrome by the UMASS morphology core, with rat anti-mouse CD68 (1:200) (AbD Serotec, clone FA-11) and Cy3-smooth muscle actin (Sigma S6198) (1:200), or with anti-ICAM-1 BBIG-I1 (1:200) or VCAM-1 BBIG-V1 (1:200) (R&D systems) and rat anti-mouse CD31 (1:400) (BD) and mounted in Prolong Gold with DAPI (Life Technologies). Images were quantified using ImageJ or Image Pro Plus Analysis Software (Media Cybernetics).

Livers were isolated and fixed in 10% formalin, paraffin embedded, and stained with hematoxylin and eosin (H&E) or frozen in OCT and stained with Oil-Red-O. Images were taken with an Axiovert 35 Zeiss microscope (Zeiss, Germany) equipped with an Axiocam CCl camera at 10x or 20x magnification.

R1- and RFP6-derived CMs were examined by immunostaining using primary antibodies to α-actinin (Sigma, 1:800, A7811) and TNNT2 (NeoMarkers, 5 μg/mL, MS295R7). Cells were fixed in 2% paraformaldehyde in PBS, washed (3x with PBS) and permeabilized (0.2% Triton X-100, Sigma). Non-specific binding was blocked with a solution of 1% bovine serum albumin (BSA) in PBS. AlexaFluor 488- or 568-conjugated goat anti-mouse IgG or AlexaFluor 568-conjugated goat anti-rabbit IgG (all Invitrogen) were used as secondary antibodies (1:1000). Nuclei were stained with Hoechst 33342 (Molecular Probes, Eugene, OR, 5 mg/mL). Images were obtained by fluorescence microscopy using a Zeiss Axiovert 35 microscope (Zeiss, West Germany) with Zeiss lenses (Plan-Neofluar, 63X/1.25 oil, 40X/1.30, 10X/0.25 and 5X/015) coupled to a SPOT Camera (Diagnostic Instruments, Inc Sterling Heights, MI). Following acquisition (SPOT Advanced 4.0.9 software), single channel files (tiff or jpeg) were composed with the assistance of Photoshop and the adjustment of only contrast or brightness.

For immunocytochemistry experiments, adult NG neuronal cultures (grown for at least 10 days in vitro) were fixed in warmed (37°C) 4% paraformaldehyde/30% sucrose mixture, and incubated at room temperature for 10 min. After fixation, the paraformaldehyde/sucrose mixture was aspirated, and 1 ml of 0.1% (vol/vol) Triton X-100 solution in PBS was added and incubated for 10 min at room temperature. Next, the coverslips were washed once gently with 0.1 M PBS, and 1 ml of 5% (wt/vol) bovine serum albumin or 10% serum in PBS was added; samples were then incubated for an hour at room temperature. Primary antibodies (polyclonal rabbit anti P2X1, P2X2, P2X3, and P2X4, or monoclonal mouse anti NeuN; 1∶1000, Cell Signaling Technology) were then diluted in the same solution, added to the coverslips, and incubated overnight in a humidified chamber at 4°C. After rinsing the coverslips three times with PBS, secondary antibodies (goat anti-rabbit fluorescein, goat-anti-mouse Cy3, 1∶1000, Sigma-Aldrich) were added and incubated at room temperature for 1 hour. The cells were then kept in the dark until analysis. Coverslips were rinsed and mounted on slides using Immuno-Fluore mounting medium (ICN Immunobiologicals). The cells were examined by epifluorescence on a Zeiss (Axiovert 35) microscope (Zeiss, Germany).

Neurons were stressed via 10 μM MG132 for the indicated times, hypoxia-reoxygenation (1% O₂ for 3 h and 4 h recovery at 5% O₂) using a hypoxia glove box (BioSpherix Xvivo System Model X3), or incubation with oligomers made from 500 nM amyloid-β (1–42) monomers (rPeptide, #1163–1) ^{133 (link)}. As mature neurons cannot be transfected, plasmids were introduced into primary cultured mouse motor neurons by intranuclear microinjection. The injectate (the plasmid in 50% Tris-ethylenediaminetetraacetic acid (EDTA), pH 7.2) was clarified by centrifugation prior to insertion into 1 mm diameter quick-fill glass capillaries (World Precision Instruments) pulled to fine tips using a Narishige PC-10 puller (Narishige International USA, Inc., NY, USA). Cultures on coverslips were bathed in Eagle’s minimum essential medium without bicarbonate, supplemented with 5 g/L glucose, and adjusted to pH 7.4 in 35 mm culture dishes on the stage of a Zeiss Axiovert 35 microscope (Carl Zeiss Microscopy, LLC, USA) and microinjected using a Transjector 5246 or a FemtoJet Transjector and a Micromanipulator 5171 (all from Eppendorf, Hamburg, Germany). Following microinjection, coverslips were placed in regular culture medium containing 0.75% Gentamicin (Gibco) and maintained at 37°C in a 5% CO₂ environment until analysis.