Goat antimouse antibody alexafluor488

Titanium sheet and diamond-coated silicon wafers
were cut into
2.2 × 2.2 cm² squares. The diamond films were terminated
with hydrogen and oxygen as described in Section 2.1.3. The substrates were sterilized in 70%
ethanol for 10 min and air-dried inside a laminar flow hood. The substrates
were adhered to the bottom of a reusable eight-well silicon insert
(flexiPERM^R, Heraeus Instruments). Cells were seeded at
the densities stated in Section 2.4.1 and cultured for 5 days. The medium
was changed every second day, and on day 5, cells were fixed with
4% paraformaldehyde for 15 min. Cells were then permeabilized with
0.2% Triton X-100, blocked with 4% BSA/4% FBS, and incubated overnight
at 8 °C with mouse antivinculin antibody (clone hVIN-1, Sigma-Aldrich).
The antivinculin antibody was detected with goat antimouse antibody-AlexaFluor
488 (Thermo Fisher Scientific). Cells were counter-stained with DAPI
and Phalloidin-Atto 565 (Sigma-Aldrich). Finally, substrates containing
the stained cells were mounted on #1.5 glass coverslips using Mowiol^R (Sigma-Aldrich) mounting media. Specimens were imaged using
a TCS SP8 confocal microscope (Leica Microsystems) equipped with hybrid
detectors, white and blue diode lasers, and a 40× immersion objective
(NA = 1.1). Whole volume images of the cells were acquired with a z-step of 0.5 μm.

Cells were fixed with 4% PFA (Thermo Fisher Scientific, Waltham, MA, USA) for 15 min at RT and then permeabilized with 0.1% TritonX-100 in sterile PBS for 10 min at RT, after blocking the samples with 5% BSA and 5% FBS in sterile PBS for 1 h at RT. Next, cells were incubated with the appropriate primary antibody overnight at 4 °C, and after several washing steps, secondary antibodies were used. The cell nuclei were stained with DAPI (Thermo Fisher Scientific). A Zeiss LSM-710 confocal microscopy system (Carl Zeiss Microscopy GmbH, Jena, Germany) was used to detect proteins of interest with a 63× objective. Images were analyzed with ZEN 3.2 (Carl Zeiss Microscopy GmbH, Jena, Germany) and Image J software (Version 1.53t) [38 (link)]. Primary antibodies were the same as we used for Western blot. Secondary antibodies used were Goat-Anti-Mouse antibody, Alexa Fluor 488, Thermo Fisher Scientific, Waltham, MA, USA; Cat# A-11029, Goat anti-Rabbit IgG, Alexa Fluor 488, Thermo Fisher Scientific, Waltham, MA, USA; Cat# A-11008, Goat-Anti-Rabbit antibody, Alexa Fluor 546, and Thermo Fisher Scientific, Waltham, MA, USA; Cat# A11035 for ICC analysis. Actin filaments were stained with CF^®543 Phalloidin (Biotium, Fremont, CA, USA; Cat: 00043). The colocalization coefficient was calculated with Image J-Ezcolocalization plugin according to this article [40 (link)].

Transformed BL21 (DE3) ΔA E. coli (4 × 10⁸ cells) was washed once with PBS. The bacterial pellet was fixed with 4% paraformaldehyde at RT for 30 min and washed twice with PBS before mixing with 100 μL of 1:200 of mouse mAb to HuscFv34 (clone 5F7) and kept at RT for 1 h. After centrifugation, the pellet was washed twice with 500 μL of FACS buffer (2% FBS and 0.02% sodium azide in PBS) and stained with 100 μL of 1:200 goat anti-mouse antibody-Alexa Fluor 488 (Thermo Fisher Scientific) on ice, in darkness for 1 h. After washing with FACS buffer, the pellet was stained with 200 μL of 1:250 of 4′,6-diamidino-2-phenylindole (DAPI) at RT in darkness for 30 min. The stained bacterial cells were washed and resuspended in 300 μL of 1% paraformaldehyde. The PEN-HuscFv34-6× His displayed on bacterial cell surface was determined by flow cytometry (BD LSRFortessa™, BD).