The cells were seeded on 8-well chamber slides (NuncTM Lab-TekTM II Chamber SlideTM System/Thermo Fisher Scientific Inc, Waltham, MA, USA). Once the slides reached 80% confluence, experimental treatments were performed for 48 h. Cell fixation was performed in 70% ice-cold methanol for 15 min in a freezer. The cells were washed three times in DPBS. Subsequently, cells were immersed in the blocking buffer (5% FBS, 0.3% Triton X-100 in DPBS 1X) for 1 h. Incubation with the primary antibody CAV-1 (sc-894, Santa Cruz Biotechnology, Dallas, TX, USA) was performed overnight (1:100, 1% BSA, 0.3% Triton X-100 in DPBS 1X). The following day, cells were washed three times with DPBS and incubated for 1.5 h with a secondary antibody (1:400, Alexa Fluor Plus® 488, goat anti-rabbit, #A32731, Thermo Fisher Scientific Inc, Waltham, MA, USA). Upon completed incubation, the final washing step was performed (three times in DPBS 1X), and the mounting medium, Fluoroshield with DAPI (F6057, Sigma, St. Louis, MO, USA), was used to preserve the fluorescence of cell specimens. Confocal fluorescence imaging was performed with the use of Olympus iXplore SpinSR ScanR (Olympus, Tokyo, Japan).
Nunc lab tektm 2 chamber slide system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Nunc™ Lab-Tek™ II Chamber Slide™ System is a multi-well cell culture slide designed for microscopic analysis. It provides a convenient platform for growing, observing, and analyzing adherent cells under a microscope.
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2 protocols using nunc lab tektm 2 chamber slide system
1
Immunofluorescence Analysis of Caveolin-1
2
Immunofluorescence Assay for Leptospira Detection
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IFA was carried out on VF and EB samples as previously described (Pinne and Haake, 2011) (link), and conducted only on those samples that were qPCR positives. After fixed with 1% formaldehyde, imprint slides were incubated for 1 h at 37 C with a primary rabbit polyclonal anti-rLipL32 antibody (1:500) or with a rabbit preimmune serum (1:500; used as negative control), followed by three washes with 3% PBS-BSA pH7.4. Finally, imprints were incubated with Alexa Fluor1 488 goat anti-rabbit IgG (H + L) (1:2,000) for 1 h at 37 C and washed three times with 3% PBS-BSA pH7.4. Two hundred microliters of each VF sample were added into each well in the precoated poly-L-lysine-chamber slide (Nunc TM Lab-Tek TM II Chamber Slide TM System) (Thermo Scientific, Waltham, MA, USA) and incubated for 1 h at 37 C. Slides were fixed with 1% formaldehyde. Incubations with primary and secondary antibody were performed as described above. Both imprints and slides were dried for 15 min and assembled using coverslips and antifade (Prolong Gold antifade) (Life Technologies, Carlsbad, CA, USA). After 24 h, the slides were sealed using colorless glaze and viewed under a microscope AxioImager m1 (Zeiss) (Thornwood, NI, USA).
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