Alexa fluor 594 conjugated goat anti rabbit

It was performed as in [43 (link)] with the following antibodies: anti-CK1α (abcam, UK), secondary Alexa-Fluor 594-conjugated goat anti-rabbit (Life technologies, Italy). Specimens were mounted in Vectashield medium with DAPI (Vector Laboratories, USA) and analyzed using Leica TCS SP5 confocal microscope, oil objective 63x with the LAS Advances Fluorescence Leica Application Suite software (Italy).

Thin smears of P. falciparum-iRBCs on glass slides were briefly dried and fixed with 4% paraformaldehyde and 0.005% glutaraldehyde in PBS for 15 min at room temperature. After rinsing with 50 mM glycine, the slides were blocked with 5% of skim milk in PBS for 30 min at 37 °C. The slides were first incubated with mouse monoclonal anti-GFP (Roche, Switzerland) and rabbit anti-EXP2 antiserum at 1:1000 dilutions or rabbit anti-SBP1 at 1:500 dilutions or rat anti-PfEMP1 1:500 dilutions at 37 °C for 1 h. Then, the slides were incubated with Alexa-Fluor 488-conjugated goat anti-mouse or Alexa-Fluor 594-conjugated goat anti-rabbit or Alexa-Fluor 594-conjugated goat anti-rat antibodies at 1:2000 (Thermo Scientific, USA) at 37 °C for 30 min. DAPI (Sigma) was used at 1 μg/ml as a counterstain of parasite nuclei. ProLong^® Diamond Antifade Mountant (Thermo Scientific, USA) was applied onto the slide to reduce quenching under the UV light. The slides were viewed with a Nikon ECLIPSE 80i microscope. The signal intensity of immunofluorescence in P. falciparum transfectants was measured with ImageJ software (1.44p; http://rsbweb.nih.gov/ij/).

Following antibodies were used for this study. AβmOC64 (ab201060, Abcam, MA, USA), Aβ 1-42 (ab10148,), BACE1 (ab63954, Abcam, MA, USA), APP (ab15272, Abcam, MA, USA) CD63 (ab216130, Abcam, MA, USA), CD9 (ab92726, Abcam, MA, USA), Flotillin (ab133497, Abcam, MA, USA), TSG101 (ab125011, Abcam, MA, USA), Alix (ab275377, Abcam, MA, USA), Calnexin (ab133615, Abcam, MA, USA), Argonuate-2 (ab186733, Abcam, MA, USA), GFAP (G3893, Sigma- Aldrich, MO, USA), β-actin (A5316, Sigma- Aldrich, MO, USA), HIF-1α (NB100-449, Novus Biological Company, CO, USA), goat anti-rabbit (sc-2004, Santa Cruz Biotechnology, TX, USA) and goat anti-mouse (sc-2005, Santa Cruz Biotechnology, TX, USA), Alexa Fluor 488 conjugated goat anti-mouse (A11001, ThermoFisher Scientific, MA, USA), Alexa Fluor 647 conjugated goat anti-mouse (A32728, ThermoFisher Scientific, MA, USA), Alexa Fluor 594 conjugated goat anti-rabbit (A11012, ThermoFisher Scientific, MA, USA).

Cells were grown on coverslips in 12-well plates (1 × 200 cells/well) overnight and then treated with corresponding chemicals that was performed as previously described. After that, cells were postfixed with 4% PFA for 20 min, then washed with PBS for three times (10 min/time), and then treated with 10% goat or donkey serum diluted in PBS-0.3% Triton X-100 for 30 min at room temperature. Next, cells were incubated with antibodies with P62 (1:500, Sigma, P0067), LC3 (1:200, Santa, sc-376404), LAMP2 (1:200, Abcam, ab25631), LC3 (1:500, Sigma, L7543), SOD1 (1:400, Abcam, ab16831), or Rab7 (1:500, Abcam, ab137029) overnight at 4 °C. The cells were washed with PBS for three times and stained with secondary antibodies, Alexa Fluor 594-conjugated goat anti-rabbit (1:1000, Thermo Fisher, #A-11037) or Alexa Fluor 647-conjugated goat anti-mouse (1:1000, Thermo Fisher; #A-21236), for 1 h at room temperature, then stained nuclei with DAPI Fluoromount-G (Southern Biotech). Finally, imaged the cells by confocal microscopy.

The iPSC clones were confirmed as IPSCs with a battery of rabbit anti-mouse IPSC markers including Oct3/4, Sox2, c-Myc, mKlf4, Nestin and SSEA-1 (Thermo Fisher Scientific, Waltham, MA, USA). The secondary antibody was an Alexa Fluor 594-conjugated goat anti-rabbit (Thermo Fisher Scientific), all used in accordance with the manufacturer’s conditions. To immobilize the iPSC clones, glass-bottom dishes were coated with Cell-TEK adhesive. The adherent iPSC was then fixed with 4% paraformaldehyde, after permeabilizing with TX-100 and blocking with normal goat serum. The iPSC clones were then incubated with the primary antibodies, washed, and incubated with the secondary antibodies. The dishes were finally mounted with Vectorshield mounting medium with DAPI (#H-1200) (Vector Laboratories, Burlingame, CA, USA) and viewed with an Olympus Fluoview-1000 confocal scanning system under different wavelengths.

Immunofluorescence was performed following [29 (link)]. Briefly, the cells were fixed in 4% formaldehyde (PFA) for 10 min at room temperature (RT) and permeabilized with 0.2% Triton X-100 (#194854; MP Biomedicals, USA) for 10 min and 0.1% saponin (#S7900; Sigma Aldrich, Czech Republic) for 12 min. After that, the dishes were washed twice in phosphate-buffered saline for 15 min. We used 1% bovine serum albumin (BSA; #A2153-506; Sigma Aldrich, Czech Republic) dissolved in 1× PBS as a blocking solution. Next, the samples were incubated in blocking solution for one hour at room temperature and then washed in 1× PBS for 15 min. For immunofluorescence analysis, the following antibodies were used: anti-ACE2 (#ab15348; Abcam, UK) and α-actinin (#A7811; Sigma Aldrich, Czech Republic). As secondary antibodies, we used the following: Alexa Fluor 594-conjugated goat anti-rabbit (#A11037; ThermoFisher Scientific, Czech Republic), Alexa Fluor 594-conjugated goat anti-mouse (#A11032; ThermoFisher Scientific, Czech Republic), Alexa Fluor 488-conjugated goat anti-rabbit (#ab150077; Abcam UK) antibodies. The negative control was considered samples incubated without primary antibodies. Cell nuclei (GC-rich sequences of DNA) were stained with 4′,6-diamidino-2-phenylindole (DAPI; Merck, Czech Republic). As a mounting medium, we used Vectashield (#H-1000, Vector Laboratories, USA).