Streptavidin alexa fluor 594

TJ permeability assays were undertaken as previously described [17] (link), [50] (link). Briefly, a solution of 10 mg/ml EZ-Link Sulfo-NHS-LC-Biotin (Pierce) in PBS containing 1 mM CaCl₂ was injected into the paw pads of P3 pups. Paw pads were incubated at room temperature for 30 minutes prior to frozen sectioning and IHC with conjugated Streptavidin Alexafluor 594 (Life Technologies, S-11227).

Tissue was then snap frozen in OCT Tissue-Tek freezing medium (Sakura). Cryostat sections of 7 µm were air dried, fixed with acetone, and washed in DPBS. Blocking was performed with 20% normal horse serum (PAA Laboratories Inc.) for 15 min at room temperature. Sections were incubated with biotin-conjugated anti-mouse IgM (clone 11–41, BD Biosciences), followed by streptavidin Alexa Fluor 594 (Life Technologies) or FITC-conjugated anti-mouse monocyte and macrophage (MOMA; Abcam). Negative controls were no primary antibody or stained with isotype-matched antibody. Images were acquired on an Olympus BX51 microscope using Micro-Manager software (Vale Laboratory).

The following antibodies were used: anti-GFP (A11122, Invitrogen); anti-IIIβ-tubulin (MMS-435P, Covance); anti-growth associated protein 43 (GAP43) (AB5220, Millipore); anti-myelin basic protein antibody (MBP) (ab7349, Abcam); anti-neurofilament H (NF-H) (AB5539, Millipore); Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody Alexa Fluor 488 (A-21202, Thermo Fisher); Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody Alexa Fluor 488 (A-21206, Thermo Fisher); Goat anti-Chicken IgY (H+L) Alexa Fluor 488 (A-11039, Thermo Fisher), Biotinylated Horse anti-rabbit IgG (BA-1000, Vector); Biotinylated Goat anti-rat IgG (BA-9400, Vector), Streptavidin-Biotinylated HRP Complex (RPN1051, GE Healthcare); and Streptavidin-Alexa Fluor 594 (S32356, Thermo Fisher).
The following drugs and reagents were used: poly-D-Lysine (P7280, Sigma); laminin (L2020, Sigma); Nystatin dihydrate (N4014, Sigma); Cholesterol Oxidase Streptomyces sp. (ChOx) (228250, Calbiochem); Methyl-β-cyclodextrin (MβCD) (C4555, Sigma); DMSO (D5879, Sigma); phalloidin – TRITC (P1951, Sigma); biocytin (B4261, Sigma); Cholera Toxin Subunit B (Recombinant) Alexa Fluor 594 (CTxB-594) (C34777, Life BioSciences).

The following antibodies were purchased from BioLegend (San Diego, CA): anti‐CD3e (145‐2C11), anti‐CD4 (RM4‐5), anti‐CD11b (M1/70), anti‐CD31 (390), anti‐CD45 (30‐F11), anti‐CD45R/B220 (RA3‐6B2), anti‐CD169 (3D6.112), anti‐F4/80 (BM8), anti‐MAdCAM‐1 (MECA‐367), anti‐Syrian hamster biotin (Poly4056) and anti‐TER119 (TER119). Anti‐CD35 (8C12) and anti‐CD16/32 (2.4G2) were purchased from BD Biosciences. Streptavidin Alexa Fluor 594, Alexa Fluor 488 and Alexa Fluor 647 were purchased from ThermoFisher Scientific. Anti‐podoplanin/GP38 (8.1.1) was purchased from the Developmental Studies Hybridoma Bank (Iowa City, IA).

Analysis of neuron anatomy was done as previously described [99 (link)]. Briefly, after filling MSNs with a biocytin-containing intercellular solution during electrophysiological analyses, brain slices were removed from the recording chamber, fixed with 4% Histofix (Roth, Germany) and subsequently stained with Streptavidin-Alexa Fluor 594 (dilution of 1:1000; ThermoFisher Scientific Inc., Waltham, MA, USA). Slices were mounted onto slides with ProLong Gold antifade reagent (ThermoFisher Scientific Inc., Waltham, MA, USA).
Proximal and distal dendrites of MSNs in the striatum were imaged with a 63× objective (Olympus, Shinjuku, Japan) on a confocal microscope (Leica SP5 LSM; Leica, Wetzlar, Germany). Subsequent blind deconvolution was performed with Amira software (ThermoFisher Scientific Inc., Waltham, MA, USA). Whole-cell images for Sholl analysis were taken with a 40× objective (Olympus, Shinjuku, Japan).
Semi-automatic spine counting of proximal and distal dendrites and tracing of dendrites of whole cells was carried out with NeuronStudio (version 0.9.92; Computational Neurobiology and Imaging Center Mount Sinai School of Medicine, New York, NY, USA). Sholl analyses of the reconstructed neurons were performed with Neurolucida (version 3.70.2; MBF Bioscience; Williston, VT, USA), with starting radius and radius increment parameter set to 10 µm.

Frozen sections 6–8 μm thick were fixed in ice‐cold acetone, rehydrated in PBS and blocked with normal horse serum before antibody application. The following antibodies were purchased from BioLegend: anti‐CD1d (1B1), anti‐CD4 (RM4‐5), anti‐CD21/35 (7E9) and anti‐CD45R/B220 (RA3‐6B2). The following antibodies were purchased from BD Biosciences: anti‐CD35 (8C12), anti‐MAdCAM‐1 (MECA‐367) and anti‐TNP (G235‐1). Anti‐CD169 (MOMA‐1) and anti‐MARCO (ED31) were purchased from Bio‐Rad (Hemel Hempstead, UK). Anti‐CD209b/SIGNR1 (eBio22D1) and phycoerythrin (PE)‐conjugated anti‐Armenian hamster IgG were purchased from eBiosciences (ThermoFischer, Loughborough, UK). Anti‐CXCL13 (polyclonal) was purchased from R&D Systems. Streptavidin Alexa Fluor 594, goat anti‐rat IgG (H+L) Alexa Fluor 594, donkey anti‐goat IgG (H+L) Alexa Fluor 647 and goat anti‐rat IgG (H+L) Alexa Fluor 488 were purchased from ThermoFisher Scientific (Waltham, MA). Dako fluorescent mounting medium (Agilent, Santa Clara, CA) was used to apply coverslips before image acquisition. A Zeiss LSM5 Pascal (Carl Zeiss, Oberkochen, Germany) upright microscope with zen software (Rochdale, UK) was used for image collection.

The DNA templates of hybridization probes for RNA fluorescent in situ hybridization (RNA-FISH) were generated by PCR (Supplementary Table S1). The FISH probes of PF3D7_1240600 and PF3D7_0617400 mRNAs were, respectively, generated and labeled with biotin or fluorescein (Biotin-High Prime and Fluorescein-High Prime kits, Roche) according to each standard manual.
For detection of var gene mRNA localization, FISH was carried out as reported with some modifications (Epp et al., 2009 (link)). Fixed parasites for RNA-FISH were prepared as described above in the immunofluorescence assay and placed on polylysine-coated adhesion microscope slides (Citoglas). After permeabilization with 0.1% TritonX-100/1 × PBS for 7 min, washing with 1 × PBS and blocking with 1%BSA/1 × PBS for 30 min, FISH was performed in hybridization buffer [50% deionized formamide (Ambion), 10% dextran sulfate (MW > 500,000, Sigma-Aldrich), 2 × SSPE (Ambion), 250 μg/ml sheared salmon sperm DNA] with 70 ng/μl of each labeled probe at 37°C overnight. Slides were then washed with 50%formamide/2 × SSC and 2 × SSC (Ambion), blocked with 5%BSA/2 × SSC for 30 min, incubated with streptavidin-Alexa Fluor 594 (Thermo) for 30 min and washed with 2 × SSC. Finally, the results of RNA-FISH for PF3D7_1240600 and PF3D7_0617400 mRNAs were recorded by Olympus FV-1200.