Axiocam digital camera

A conjugated anti-Cryptosporidium spp. (Sporoglo, Waterborne, New Orleans, LA, USA) was used in direct fluorescent-antibody staining assay according to the manufacturers’ instructions. DAPI (4′,6-diamidino-2-phenylindole) was used for nuclei identification. Slides were examined in a LSM880 Confocal Microscope (Zeiss, Oberkochen, Germany) equiped with a UV laser for the detection of DAPI (excitation at 355 nm and emission at 405 nm) and VIS (visible light) laser for the detection of sporoglo (excitation wavelength at 555 nm and emission at 561 nm). The images were taken using Ziess Axiocam digital camera. The images were processed using Carl Zeiss Zen software.

For mouse brain staining, brains were dissected and fixed in 4% paraformaldehyde for 1 hr on ice followed by immersion in 20% sucrose overnight at 4°C. Brains were then sectioned using a freezing stage Microtome (Microm KS 34, Thermo Scientific) and spread out on glass coverslips for immunostaining. The antibodies were used at the following concentrations: mouse anti-AnkG (N106/36, N106/20) (UC Davis/NIH), 1:200; rabbit anti-ATF3 (Santa Cruz) (RRID:AB_2058590), 1:500; chicken anti-GFP (ab13970, abcam) (RRID:AB_300798), 1:1000; rabbit anti-Iba1 (019–19741, Wako), 1:250; chicken anti-GFAP (ab4674, abcam) (RRID:AB_304558), 1:500; NeuroTrace (Thermo Scientific), 1:200; Alexa 488-, Cy3-, or Cy5-conjugated secondary antibodies (Jackson ImmunoResearch), 1:250. Samples were then mounted in Vectashield (Vector Laboratories) before being analyzed under a confocal microscope. The images were obtained with an Axio-imager Z1 microscope (Carl Zeiss) fitted with an AxioCam digital camera (Carl Zeiss). The images were analyzed by ZEN 2012 (Carl Zeiss).

To further characterize the morphological structure of aggregated serum albumins, we used the specific amorphous aggregate binding dye ANS, which was visualized by fluorescence microscopy. Serum albumin aggregates obtained after incubation time points were incubated with 100 μM of ANS dye for 30 minutes at room temperature and then transferred to a glass slide. Now, the samples were imaged using fluorescence microscopy (Axio, HBU 50/AC; Zeiss, Gottingen, Germany). The images were acquired using a Carl Zeiss Imager M2 microscope (20x objective magnifications) equipped with AxioCam digital camera. The microscope is controlled by ZEN2010 image processing software. For precise imaging, Z-stacking was done in the FITC channel.

To assess the levels of intracellular Zn²⁺, cells were incubated with 2 µM FluoZin-3 AM (Kd for Zn²⁺, about 15 nM) (Molecular Probes, Eugene, OR, USA) for 30 min and washed with fresh culture medium (Gee et al., 2002 (link)). FluoZin-3 reactive cells were examined or photographed under consistent imaging conditions with an inverted fluorescence microscope (Axio Observer.Z1) using a FITC filter (beam splitter, 495 nm; excitation, 450–490 nm; emission, 500–550 nm) equipped with self-adjusting lamps and an AxioCam digital camera (Carl Zeiss).
To quantify the level of intracellular Zn²⁺, we took the photographs (magnification, 100×) from three spots randomly selected from each culture well, and measured the mean intensity of FluoZin-3-fluorescence in neurons using ImagePro Plus software (Media Cybernetics, Silver Spring, MD, USA). After subtracting the background intensity (which was determined by assessing areas without cells), the average intensity of FluoZin-3-fluorescence per neuron was reported as the level of intracellular Zn²⁺.

Five-μm sections were dewaxed and rehydrated. After quenching endogenous peroxidase, achieving antigen retrieval and blocking nonspecific binding sites, sections were incubated with the anti-human VEGF-A mouse monoclonal antibody (Beckton Dickinson), at a concentration of 5 μg/ml, anti-mouse PECAM/CD31 rabbit polyclonal antibody (Abcam), at a concentration of 40 μg/ml and anti-mouse CD-54/ICAM-1 hamster monoclonal antibody (BD Pharmingen) at a concentration of 1.25 μg/ml. Secondary biotinylated monoclonal Abs and staining kits were obtained from Vector Laboratories. Immunoreactivity was visualized with peroxidase reaction using 3-amino-9-ethylcarbazole (AEC, Vector Laboratories, Burlingame, CA) in H₂O₂ and specimen counterstained with hematoxylin. As a negative control, primary Abs were omitted or replaced with an irrelevant isotype-matched mAb. Stained sections were analyzed with the AxioCam digital camera attached to the Axioplan 2 microscope (Carl Zeiss AG, Oberkochen, Germany). VEGF-A staining intensity was evaluated by a semiquantitative, four-stage scoring system, ranging from negative (0) to strong immunoreactivity (4+). Positive cells for ICAM-1 and CD31 were directly counted. Every quantification was performed on three fields per sample by two independent observers, blinded to the status of the specimens.