Alexa fluor 594 streptavidin

BDA and CTB fluorescence staining was used to demonstrate anterogradely labeled fibers and retrogradely labeled neurons.
For CTB fluorescence staining, slices were washed with PBS before (3 × 10 min) and after (1 × 5 min) incubation in 10% normal goat serum (30 min; 1:10 in PBS). Subsequently, they were incubated with polyclonal rabbit anti-CTB antibody (Abcam, RRID: AB_34992; 1:1,000 in PBS) for 12–14 h at 4℃. Then, the slices were washed in PBS (3 × 10 min) and incubated with the secondary antibody Alexa Fluor 488-conjugated AffiniPure Goat Anti-Rabbit IgG (Wuhan service biotechnology; 1:200 in PBS) for 1 h at 37℃. Finally, the slices were washed in PBS (3 × 10 min), mounted on glass slides, and covered using a coverslip with fluoromount (Wuhan service biotechnology, Wuhan, China).
For BDA fluorescence staining, after the slices were incubated for 30 min in 10% normal goat serum, they were incubated with Alexa Fluor 594 streptavidin (BioLegend Way San Diego, CA; 1:200 in PBS) for 50 min at 37℃. Finally, the slices were washed in PBS (3 × 10 min), mounted on glass slides, and covered using a coverslip with fluoromount.

Fluorochrome-conjugated isotype against human CD3 (APC/Cy7/FITC, UCHT1), CD4 (Pacific Blue, OKT4), CD8 (PE, SK1, 344706), CD45RA (PE/Cy7, HI100), CCR7(APC, G043H7), CD123 (FITC, 6H6), and streptavidin-Alexa Fluor 594 were purchased from Biolegend (San Diego, CA, USA). The antibody of Biotin-SP-conjugated AffiniPure Goat Anti-Mouse IgG, F(ab′)2 Fragment Specific was obtained from Jackson Immunoresearch. Samples were analyzed using the ACEA NovoCyte flow cytometer and the Novoexpress software (ACEABIO). For detection of CAR surface expression, T cells were incubated with Biotin-conjugated Anti-Mouse F(ab′)2 for 20 min and followed by two washes and stained with streptavidin-Alexa Fluor 594 and FITC labeled CD3 for another 20 min at 4°C. Based on the phenotype and functions, T cells would be divided into four different subsets: naïve T cell (T_naive, CCR7⁺ CD45RA⁺), central memory T cell (T_CM, CCR7⁺ CD45RA⁻), effector memory T cell (T_EM, CCR7⁻CD45RA⁻) and CD45RA⁺ effector memory T cell (T_EMRA, CCR7⁻CD45RA⁺).

Antibodies for immunofluorescence were purchased from the following sources: F4/80 (0.2 μg/ml, BM8, OriGene, Rockville, MD, USA), TNF-α (2.5 μg/ml, MP6-XT3, eBioscience, San Diego, CA, USA), IL-10 (2.5 μg/ml, JES5-2A5, eBioscience), fibronectin (0.7 μg/ml, Polyclonal, invitrogen, Carlsbad, CA, USA), and collagen IV (2.5 μg/ml, Polyclonal, Invitrogen). Secondary antibody were goat anti-rat IgG Alexa Fluor® 488 (2 μg/ml, BioLegend, San Diego, CA, USA), streptavidin Alexa Fluor® 594 (0.5 μg/ml, BioLegend), goat anti-rabbit IgG Alexa Fluor® 488 (2 μg/ml, Invitrogen).
The following antibodies (BioLegend) were used for flow cytometry: APC anti-mouse F4/80 (2.5 μg/ml, BM8), PE/Cy7 anti-mouse CD86 (1.25 μg/ml, GL-1), PE anti-mouse CD206 (1.25 μg/ml, C068C2), PE/Cy7 anti-mouse TNF-α (5 μg/ml, MP6-XT2), and Alexa Fluor® 488 anti-mouse IL-10 (12.5 μg/ml, JES5-16E3).