Alexa fluor 488 conjugated donkey anti goat igg

Tissue sections (40 μm) and cultured cells were fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton X-100. The samples were then blocked with 10% donkey serum and probed with anti-CD31 (RD, AF3628), anti-Laminin (Abcam, ab11575), anti-VE-cadherin (Santa Cruz, sc-9989), anti-occludin (Proteintech, 66,378-1-Ig), anti-claudin 5 (Thermo, 35-2500), anti-NRF1 (CST, 46743S), anti-CAV-1 (CST, 3267), anti-ZO-1 (Proteintech, 21,773-1-AP) or anti-LC3 (CST, 12741S) antibodies. The binding of primary antibodies was visualized with either Alexa Fluor 555-conjugated donkey anti-rabbit IgG (Thermo, A31572), Alexa Fluor 647-conjugated donkey anti-mouse IgG (Thermo, A32787), or Alexa Fluor 488-conjugated donkey anti-goat IgG (Abcam, ab150133). The samples were then counterstained with DAPI (Thermo) and imaged using a Leica SP8 confocal microscope.

The cell monolayers were infected with L. interrogans strain Lai at an MOI₁₀₀ for 1, 2, 4, 8, 12 or 24 hr as above. Using rat anti-strain Lai-IgG, rabbit anti-Rab11-IgG (Cell Signaling) or goat anti-Sec15 or Sec-3-IgG (Santa Crus) as the primary antibody as well as AlexaFluor594-conjugated donkey anti-rat-IgG, AlexaFluor405-conjugated donkey anti-rabbit-IgG or AlexaFluor488-conjugated donkey anti-goat-IgG (Abcam) as the second antibody, Lep-vesicle-Rab11-Sec15/Sec3 co-localization (Lep-vesicle-recycling endosome-exocyst complexes) was detected by confocal microscopy and the co-localization percentages and white FI reflecting the co-localization were determined as above. Uninfected cells were used as controls.

Organs were placed in optimal cutting temperature compound (OCT, Sakura Finetek, Torrance, CA, USA) and flash frozen in liquid nitrogen. Sections were sliced at 6 mm thickness and fixed in 5% paraformaldehyde at room temperature for 15 min. Slides were then blocked and permeabilized in 5% non-fat milk in 0.05% Tween-20 PBS overnight at 4 °C. After washing, the slides were incubated with the primary antibody diluted in 2% non-fat milk/PBS for 1 h, followed by the secondary antibody diluted in 2% non-fat milk/PBS for 1 h. Slides were then mounted using VECTASHIELD Antifade Mounting Medium with DAPI (Vector Labs, Burlingame, CA, USA) before visualization using fluorescent filters under a EVOS M5000 microscope (Invitrogen). Images were overlaid using ImageJ software (NIH, Bethesda, MD, USA). The following antibodies were used on 4% para-formaldehyde-fixed OCT sections of monkey organ/tissue: anti-HAdV3 fiber knob mAb- clone 2-1; goat-α-human DSG2 (AF947, R&D Systems), mouse-α-CD163 (Biolegend #333602) with the secondaries as follows: Alexa-Fluor 594 conjugated chicken anti-rabbit IgG (Invitrogen A-21442), Alexa Fluor 488-conjugated donkey anti-goat IgG (Abcam #6881), and Alexa Fluor 488-conjugated goat anti-mouse IgG (Biolegend #405319).

The cell monolayers were infected with L. interrogans strain Lai at an MOI₁₀₀ for 1, 2, 4, 8, 12 or 24 hr as above. Using rat anti-strain Lai-IgG, goat anti-Sec15-IgG (Santa Crus), rabbit anti-VAMP2 (Cell Signaling) or SYN1-IgG (Abcam) as the primary antibody as well as AlexaFluor594-conjugated donkey anti-rat-IgG, AlexaFluor488-conjugated donkey anti-goat-IgG or AlexaFluor405-conjugated donkey anti-rabbit-IgG (Abcam) as the second antibody, the Lep-vesicle-Sec15-VAMP2 or SYN1 co-localization (Lep-vesicle-recycling endosome-exocyst-SNARE complexes) were detected by confocal microscopy and the co-localization percentages and white FI reflecting the co-localization were determined as above. Uninfected cells were used as controls.