Axiovert 200 microscope

Macrophages infected with T. gondii were fixed in 4% paraformaldehyde in PBS for at least 1 h, at room temperature. After washing with PBS, cells were permeabilized with 0.1% Saponin for 30 min at room temperature and with 100% acetone for 10 min at -20°C. Samples were blocked in 3% BSA/PBS blocker buffer for 40 min. Infected cells were then incubated 1 h with anti-SAG-1 (1:1000) and anti-LAMP-1-PE (0.2 mg/10⁶ cells) antibodies in blocker buffer. Then, samples were washed in 1% BSA/PBS for 15 min 3 times, incubated 30 min with blocker buffer, and labeled with goat anti-rabbit-Alexa Fluor 488 secondary antibodies (1:1000, for 1 h, at room temperature). Coverslips were washed gently with distillated water, mounted onto slides using Vectashield (Vector Labs. USA), and samples were analyzed in an Axiovert 200 microscope with an ApoTome fluorescence module (Carl Zeiss, Germany).
To evaluate phagolysosomal fusion during T. gondi infection, peritoneal macrophages were infected for 2 h as described above, incubated for 30 minutes with 100 nM of Lysotracker Red (Life Technologies, USA), and then fixed in 4% paraformaldehyde in PBS for 30 minutes. Coverslips were washed gently in distillated water, mounted onto microscope slides using Vectashield, and observed in an Axiovert 200 microscope with an ApoTome fluorescence module (Carl Zeiss, West Germany).

For Rap1-GFP foci imaging, cell were grown to the 5x10⁶ cells/ml at 34°C for 2 hours in synthetic complete (SC) media. Images were captured immediately in 21 Z-stacks of 0.2 μm using Zeiss Axiovert 200 microscope. GFP foci per nucleus were manually counted as a representation for telomere foci. For Sir4 immunofluorescence, cell cultures were grown to the 5x10⁶ cells/ml at 34°C for 2 hours in synthetic complete (SC) media. Cells were immediately fixed using 3.7% formaldehyde and spheroplasted in SK (0.1M KPO₄/1.2M sorbitol) buffer containing 0.4 mg/ml Zymolase (US, Biological). Spheroplasted cells were fixed on poly-lysine coated coverslips as described previously [84 (link)]. Coverslips were blocked in 1% BSA in PBS for 1 hour, then incubated with primary (αMyc, ab9106-100) followed by secondary (Alexa 488; Molecular Probes, Invitrogen) antibodies each for 30 minutes. Coverslips were mounted on microscope slides using vectashield-containing DAPI (Molecular Probes, Invitrogen). Images were taken in 21 Z-stacks of 0.2 μm using Zeiss Axiovert 200 microscope and Z-stack images were flattened and presented in the figures. ImageJ (NIH, USA) was used for adjusting background in both live and immunofluorescence imaging methods.

Immunohistochemical analysis of human breast cancer tissues in TMA was performed according to the avidin-biotinylated-HRP complex (ABC) method using an Elite ABC kit (Vector Laboratories, Burlingame, CA, USA). The TMA slides were transferred to a xylene chamber and dipped in a graded ethanol series. After antigen retrieval in 10 mM sodium citrate buffer (pH 6.0) for 15 min, the slides were transferred to 3% hydrogen peroxide in methanol to quench the endogenous peroxidase activity. To block non-specific binding sites, slides were incubated in diluted normal goat serum at 24°C for 30 min. The slides were then incubated overnight at 4°C with anti-GLI1 (ab217326, 1:1000). The slides were washed twice with TBS and incubated with biotinylated goat anti-rabbit IgG (1:200) for 30 min at 24°C. After rinsing the slides in TBS, they were incubated with ABC reagent for 30 min. Color development was achieved by applying DAB solution for 2 min. After washing in distilled water, the slides were counterstained with hematoxylin, dehydrated with ethanol and xylene, and coverslipped using a mounting solution. Images were captured using a Zeiss Axiovert 200 microscope (Zeiss, Germany) equipped with a PAXCAM microscope camera.

5 × 10² control and Kaiso-depleted MDA-231 and Hs578T cells were cultured in 60 mm dishes in duplicate and allowed to grow and form colonies for 10–14 days. For the soft agar assays, 5 × 10⁴ control and Kaiso-depleted MDA-231 and Hs578T cells were cultured in 0.3% Agarose in 60 mm dishes, and allowed to grow and form colonies for 10 days. After the incubation period, colonies were stained with 0.5 and 0.05% Gentian Violet diluted in methanol for the colony formation and soft agar assays respectively. Images of colonies from the colony formation assay were obtained by using a Canon digital camera and then colonies were counted manually. For the soft agar assay, 10 × images of colonies were obtained using the Zeiss Axiovert 200 microscope (Carl Zeiss Canada Ltd., ON, Canada), and then counted using the ImageJ software. Graphical representation of counts (colony numbers) was achieved using GraphPad Prism software.

Immunocytochemistry was performed as previously reported^{62 (link),63 (link)}. Briefly, the mTCFs were fixed in 4% paraformaldehyde (PFA) at room temperature for 10 min, and the human corneal epithelial cells were fixed with cold methanol at −20 °C for 30 min. After washing with DPBS, they were incubated with blocking buffer (10% goat serum, 0.5% gelatin, 3% BSA and 0.2% Tween 20 in DPBS) for 30 min and reacted with primary antibodies against FSP-1 (07–2274; Millipore, USA) or K12 keratin (sc-17098; Santa Cruz Biotechnology, USA) at 4 °C overnight. The cells were washed three times with DPBS containing 0.2% Tween 20 and incubated with an Alexa 488 secondary antibody (Life Technologies Inc., USA) at room temperature for 1 h. FSP-1 and K12 keratin signals were observed using a Zeiss Axiovert 200 microscope (Carl Zeiss, Germany).

EC cell staining was performed following the procedure of Masson-Fontana silver staining with slight modifications. Briefly, the deparaffined and rehydrated sections were incubated with 5% ammoniacal silver solution in a dark humidified chamber for 4 h at room temperature, followed by a 2-h incubation at 56 °C and an overnight incubation at room temperature. Slides were visualized utilizing the Zeiss Axiovert 200 microscope (Zeiss GmbH). The appearance of brown to black silver precipitate in cytoplasm of EC cells was counted as a positive reaction.