Brains were post-fixed with 4% PFA for 2 h at RT and infiltrated with increasing concentrations of sucrose as previously described [12 ]. Brains were divided into two parts, right hemispheres were coronally sectioned and left hemispheres were sagittally sectioned at 50 µm using a Leica VT1200 vibratome (Leica Microsystems, Wetzlar, Germany). Brain free-floating sections were permeabilized with 0.5 % Triton X-100 in PBS and incubated in 1:300 NeuroTrace 640/660 deep-red fluorescent Nissl stain (Molecular Probes, Eugene, OR, USA), in PBS containing 0.5 % Triton X-100 for 4 h. Nuclei were counter-stained with 0.5 mg/mL DAPI (Thermo Fisher Scientific, Rockford, IL, USA) and mounted in Vectashield Vibrance antifade mounting media (Vector Laboratories, Burlingame, CA, USA). Sections were analyzed with a Leica SP8-LSCM confocal microscope (Leica Microsystems, Wetzlar, Germany) with a 20X objective and 0.5 μm z steps. Z-stacks were generated with the ImageJ software [20 ].
Vectashield vibrance antifade mounting media
Manufactured by Vector Laboratories
Sourced in United States
Vectashield Vibrance is an antifade mounting media used to preserve the fluorescence of labeled samples. It is designed to reduce photobleaching and maintain the integrity of fluorescent signals during microscopic imaging.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (8 mentions)
Triton x 100, by Merck Group (2 mentions)
Lsm900 microscope, by Zeiss (2 mentions)
Airyscan 2, by Zeiss (2 mentions)
Vt1200 vibratome, by Leica (1 mentions)
Sp8 lscm confocal microscope, by Leica (1 mentions)
Glass slide, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 donkey anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 donkey anti rat igg, by Thermo Fisher Scientific (1 mentions)
Anti laminin gamma 1 a5, by Novus Biologicals (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Cas block histochemical reagent, by Thermo Fisher Scientific (1 mentions)
Application suite advanced fluorescence software, by Leica (1 mentions)
Alexa fluor 488 conjugated anti rabbit igg secondary antibody, by Thermo Fisher Scientific (1 mentions)
Texas red x phalloidin, by Thermo Fisher Scientific (1 mentions)
Formaldehyde solution, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Confocal microscope, by Leica (1 mentions)
Goat serum, by Thermo Fisher Scientific (1 mentions)
7 protocols using vectashield vibrance antifade mounting media
1
Detailed Fluorescent Labeling of Brain Sections
2
Immunolabeling of Cryosectioned Tissues
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Cryosections mounted on glass slides (Fisher Scientific) were blocked with 3 % bovine serum albumin (BSA), 2 % donkey serum, and 0.5 % Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 1 h at room temperature. The following antibodies were used (all from Thermo Fisher Scientific, Rockford, IL, USA): anti-myosin 4 monoclonal (MF20, 1:200 dilution), Alexa Fluor 488 phalloidin (1:200), Alexa Fluor 488 donkey anti-mouse IgG (1:1000), and Alexa Fluor 647 donkey anti-rat IgG (1:1000). Anti-laminin gamma 1 (A5, Novus Biologicals, Centennial, CO, USA) was used at a 1:400 dilution. Cell nuclei were counterstained with DAPI (Thermo Fisher Scientific) and mounted using Vectashield® Vibrance antifade mounting media (Vector Laboratories, Newark, CA, USA).
3
Immunofluorescence Assay for Parasite Analysis
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For IFA, parasites were smeared onto slides and air-dried. The entire process of IFA was done in a humid chamber. The parasites were fixed with 4% (v/v) paraformaldehyde (PFA) in 1x PBS for 10 mins and rinsed quickly with 1x PBS. The fixed parasites were permeabilized with 0.1% Triton X-100 in PBS for 10 mins at room temperature (RT) and then washed three times with 1x PBS for 3 mins. The parasites were blocked with a 3% (w/v) BSA in PBS for 1h at RT. The smear was stained with respective primary antibodies (mouse α-V5 1:500, rat α-HA 1:250, mouse α-Centrin 1:500, rabbit α-V5 1:500) for 1h at RT or overnight at 4°C, followed by washing three times with 1x PBS for 5 mins. The samples were incubated with fluorescently labeled secondary antibodies (1:1000) for 30–45 mins at RT and washed thrice with 1x PBS for 5 mins to remove excess unbound antibodies. The DNA content of the parasites was stained with Hoechst 33342 (1:5000) in 1x PBS for 20 mins at RT and then quickly rinsed with 1x PBS. The parasites were mounted in Vectashield Vibrance antifade mounting media (Vector Laboratories Inc. H-1700) with coverslips and stored at 4°C until imaging. The z-stacked Images were acquired on a Zeiss LSM900 microscope with Airyscan 2 with 63X objective and analyzed using FIJI software.
4
Immunofluorescence Assay for Parasite Visualization
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For IFA, parasites were smeared onto slides and air dried. The entire process of IFA was done in a humid chamber. The parasites were fixed with 4% (vol/vol) paraformaldehyde (PFA) in 1× PBS for 10 mins and rinsed quickly with 1× PBS. The fixed parasites were permeabilized with 0.1% Triton X-100 in PBS for 10 mins at RT and then washed three times with 1× PBS for 3 mins. The parasites were blocked with a 3% (wt/vol) BSA in PBS for 1 hour at RT. The smear was stained with respective primary antibodies (mouse α-V5 1:500, rat α-HA 1:250, mouse α-Centrin 1:500, and rabbit α-V5 1:500) for 1 hour at RT or overnight at 4°C, followed by washing three times with 1× PBS for 5 mins. The samples were incubated with fluorescently labeled secondary antibodies (1:1,000) for 30–45 mins at RT and washed thrice with 1× PBS for 5 mins to remove excess unbound antibodies. The DNA content of the parasites was stained with Hoechst 33342 (1:5,000) in 1× PBS for 20 mins at RT and then quickly rinsed with 1× PBS. The parasites were mounted in Vectashield Vibrance Antifade Mounting Media (Vector Laboratories Inc., H-1700) with coverslips and stored at 4°C until imaging. The z-stacked Images were acquired on a Zeiss LSM900 microscope with Airyscan 2 with 63× objective and analyzed using FIJI software.
5
Immunofluorescent Localization of NF-κB p65
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BMDMs were cultured on Poly-D-Lysine-coated (GIBCO, Grand Island, NY, USA) cover glasses in 24-well plates at a density of 2 × 10⁵ cells/well, fixed in 3.7% formaldehyde solution (Sigma-Aldrich) for 15 min, washed with PBS and permeabilized with 0.05% Triton-X-100 (Sigma-Aldrich) for 10 min, then blocked with CAS-Block Histochemical Reagent (Invitrogen) for 30 min, all at room temperature. Subsequently, the cells were incubated with p65 primary antibody (1:500; abcam, Cambridge, UK) overnight at 4 °C, followed by incubation with an Alexa Fluor® 488 conjugated anti-Rabbit IgG Secondary Antibody (Invitrogen) in dark at room temperature for 1 h. Cells were then incubated in the dark with DAPI (1:1000; Sigma-Aldrich) and Texas Red™-X Phalloidin (Invitrogen) for 10 min. After washing with PBS, cover glasses were mounted with VECTASHIELD Vibrance Antifade Mounting Media (VECTOR, Burlingame, CA, USA). Images were recorded using a Leica confocal microscope (Leica Microsystems, Germany) and the Leica Application Suite Advanced Fluorescence software.
6
Immunofluorescence Staining of Frozen Tissues
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Sections (10 μm) of OCT‐embedded frozen tissues were fixed in 10% formalin then permeabilised with .1% Triton‐X for 10 min and blocked for 1 h at room temperature with 5% bovine serum albumin (BSA; MACS BSA stock solution, Miltenyi Biotec, 130‐091‐376) and 10% goat serum (Gibco, 16210‐072) in Dulecco's Phosphate Buffered Saline (DPBS;Gibco, 14190144). Slides were incubated with primary antibodies (1:100) overnight at 4°C (mouse anti‐human CD11c [Invitrogen, 14‐0116‐82], mouse anti‐human CD11b [Invitrogen, 14‐0118‐82], rabbit polyclonal SEMA4A [Invitrogen, PA5‐101258] and rabbit anti‐human TRIM29 [Invitrogen, 703612]). Tissues were incubated with secondary antibody for 1 h at room temperature (Alexa Fluor 488 goat anti‐rabbit [Invitrogen, A‐11008]; Alexa Fluor 647 goat anti‐mouse [Invitrogen, A‐21235]). Vector TrueVIEW Autofluorescence Quenching Kit (Vector Laboratories, SP‐8400‐15) was prepared per manufacturer's instructions and added for 5 min. Then, the slide was stained with DAPI (Thermo Scientific, 62248). Vectashield Vibrance Antifade Mounting Media (Vector Laboratories, H‐1700) was used to mount coverslips. All antibodies were diluted in 1% BSA and 10% goat serum in DPBS. Slides were washed with DPBS between steps. Imaging was performed on a Nikon A1R confocal microscope using Nikon A1R software with 60× zoom with oil immersion.
7
Chromogenic IHC and IF Staining
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Tissues to be evaluated by chromogenic IHC detection of mCherry were counterstained with hematoxylin. All staining procedures were performed using a Leica Bond RX Automated Stainer (Leica Microsystems, Wetzlar, Germany) by HistoWiz Inc. IF staining of brain and spinal cord sections was performed as previously described [33 (link)]. Briefly, cryosections were incubated with primary antibodies overnight at 4 °C, followed by incubation with the appropriate secondary antibodies for 1 h at RT (Supplementary Table 1 ). Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific) and mounted with Vectashield® Vibrance antifade mounting media (Vector Laboratories, Newark, CA, USA).
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