Anti human igg alexa fluor 488

For indirect immunofluorescence the cells were seeded on cover slips at density of 20x10³ cells/well, overnight. When necessary, the cells were incubated during 24-48h with 2mg/ml Beva in DMEM 0.5% FBS. Then, cells were fixed and permeabilized in methanol during 20min. After blocking with 5% bovine serum albumin (BSA) for 30min, cells were incubated overnight at room temperature with the primary antibodies. Then, 1h of incubation with the secondary antibody anti-Human IgG-Alexa Fluor 488 (1:500 dilution, A11013, Invitrogen) in 5% BSA was performed for Beva; anti-rabbit-Alexa Fluor 568 (1:500 dilution, A11011, Invitrogen) in 5% BSA was performed for VEGF; anti-rabbit-Alexa Fluor 488 (1:500 dilution, A11008, Invitrogen) in 5% BSA was performed for MCT4, MCT1 and GLUT-1 and anti-mouse-Alexa Fluor 594 antibody (1:250 dilution, A11032, Invitrogen) for CD147, HKII, LDHA and CAXI. Additionally, to see the internalization of Beva, the cells exposed to Beva during 24h, we directly incubated with an anti-Human IgG-Alexa Fluor 488 (1:500 dilution, A11013, Invitrogen) after fixation and blocking.
Finally, after washing in PBS, cells were mounted in Vectashield Mounting Media with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories) and images were obtained with a fluorescence microscope (Olympus IX81), using the Cell P software.

Crf+ strain conidia were seeded at 1.05 × 10⁴ spores per well in Lab-Tek 8-well chamber slide (Nalge Nunc International, Villebon-sur-Yvette, France) in 300 μL RMPI medium, and incubated at 35°C for 8, 10, 14, or 24 h. At the end of the incubation, for each well, culture medium was removed and 300 μL ice cold methanol were added for 10 min. A blocking step was performed using 300 μL of a PBS solution containing 3% (w/v) BSA (Sigma-Aldrich), 2% (w/v) skimmed milk powder (Régilait, Macon, France) and 0.05% (v/v) Tween 20 (Thermo Fisher Scientific) for 10 min. Two hundred microliters of either scFv-Fc anti-Crf MS112-IIB1 antibody or isotype control diluted at 2 μg/mL in the previous solution without Tween 20 were incubated during 1 h at RT. Secondary antibody (anti-human IgG Alexa Fluor 488, Thermo Fisher Scientific) was added at 1:200 for 1 h at RT. Cell nuclei were stained with 1:1,000 Hoechst (Interchim, Montluçon, France) solution, before observing the slide using confocal microscopy (Olympus FV500).

Chromosomes were incubated overnight at 4 °C with primary antibody in a concentration of 5 µg ml⁻¹ and were subsequently diluted fivefold in PA buffer and stored for 1 h at 4 °C. Next, chromosomes were incubated with secondary antibody in a concentration of 5 µg ml⁻¹ for 1 h at room temperature. After addition of PA buffer to dilute the sample again by fivefold, chromosomes were stored for 30 min at 4 °C. To remove excess antibody, chromosomes were centrifuged at 750g for 5 min on a 20 µl glycerol cushion. The supernatant was then removed, leaving around 100 µl chromosome solution that could be used for imaging. Primary antibodies were anti-NCAPH (1:100, HPA002647, Sigma Aldrich), CREST anti-sera (1:200 HCT-0100, Immunovision), anti-TRF2 (1:100, sc-9143, Santa Cruz), anti-H3S10 (1:400, 06-570, Sigma-Aldrich) and anti-H3-Alexa Fluor 647 (1:200, 15930862, Thermo Fisher Scientific). Secondary antibodies were anti-rabbit IgG-Alexa Fluor 647 (1:500, A-21244, Thermo Fisher Scientific), anti-rabbit IgG-Alexa Fluor 568 (1:500, A-11011, Thermo Fisher Scientific) and anti-human IgG-Alexa Fluor 488 (1:500, A-11013, Thermo Fisher Scientific). Biotinylated TRF1 was detected using streptavidin–Alexa Fluor 568 (1:200, S11226, Invitrogen).