Longitudinal optic nerve sections were washed 2× in PBS, blocked in PBS-T-BSA for 1 h, and incubated with primary GAP43 antibody overnight (Table 1 ). Sections were then washed 3× in PBS and incubated with Alexa 488 donkey anti-mouse IgG (Invitrogen) for 1 h at room temperature before washing 3× in PBS and mounting in Vectashield fluorescent mounting medium (Vector Laboratories). Scarring at the ONT site was monitored by exposing GAP43-stained sections to GFAP and laminin antibodies and processing as above.
Vectashield fluorescent mounting medium
Manufactured by Vector Laboratories
Sourced in United States, United Kingdom
Vectashield is a fluorescent mounting medium designed for use with immunofluorescence and in situ hybridization techniques. It is formulated to preserve fluorescent signals and prevent fading.
Automatically generated - may contain errors
Lab products found in correlation
C2 confocal laser scanning microscope system, by Nikon (5 mentions)
Hoechst 33342, by Merck Group (5 mentions)
Cytofix cytoperm, by BD (4 mentions)
Ab124332, by Abcam (3 mentions)
Shandon cryomatrix, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 568 secondary antibody, by Thermo Fisher Scientific (2 mentions)
Sc 53015, by Santa Cruz Biotechnology (2 mentions)
Epifluorescent microscope, by Zeiss (2 mentions)
Normal goat serum (ngs), by Merck Group (2 mentions)
Lsm 700, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Alexa 488 donkey anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Ab182567, by Abcam (1 mentions)
Hcx pl apo cs, by Leica (1 mentions)
Cd144, by BD (1 mentions)
Axioscan microscope, by Zeiss (1 mentions)
Alexa fluor 568 donkey anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Axioskop2 mot plus wide field fluorescence microscope, by Zeiss (1 mentions)
Axiovision 4, by Zeiss (1 mentions)
23 protocols using vectashield fluorescent mounting medium
1
Visualizing Optic Nerve Regeneration
2
Immunofluorescence Analysis of IL-24 and IL-20RB in Celiac Disease
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Frozen duodenal biopsy samples derived from controls and children with CD were embedded into Shandon cryomatrix (ThermoElectron Co., Madison, WI, USA) and cut into 5 μm sections. Samples were incubated with primary antibodies specific for human IL-24 (ab182567; rabbit, 1:100, Abcam, Cambridge, United Kingdom) or IL-20RB (ab124332; rabbit, 1:100, Abcam) for 1 h at room temperature (RT). After repeated washing, slides were incubated with the corresponding Alexa Fluor 568 secondary antibody (1:200 anti-rabbit, Invitrogen) for 30 min at RT in the dark and counterstained with Hoechst 33342 (1:2000, Sigma-Aldrich). Finally, sections were coverslipped with Vectashield fluorescent mounting medium (Vector Laboratories, Burlingame, Calif., USA). Appropriate controls were performed by omitting the primary antibody to assure specificity and to avoid autofluorescence. Sections were analyzed with a Nikon C2 confocal laser scanning microscope system (Nikon, Minato, Tokyo, Japan).
3
Immunofluorescence Staining of PARK7 and IL-17R
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HT-29 cells were seeded in chambers and cultured for 24 h at 37 °C. After repeated washing, cells were permeabilized with Cytofix/Cytoperm (BD Pharmingen, San Diego, CA, USA) for 15 min at room temperature (RT), and were washed again. Human frozen colon biopsy samples were embedded into Shandon cryomatrix (ThermoElectron Co., Madison, WI, USA) and cut into 5 μm sections. Thereafter cells or tissue sections were incubated with rabbit anti-human PARK7 or IL-17R polyclonal IgG primary antibody (Abcam, Cambridge, United Kingdom) (1:200) for 1 h at RT. Cells were washed with WashPerm solution, and tissue sections with PBS, thereafter incubated with Alexa Fluor 488 or 568 labelled chicken anti-rabbit IgG secondary antibody (1:200, Invitrogen, Life Technologies, Carlsbad, CA, USA) for 30 min at RT in the dark and then counterstained with Hoechst 33342 (1:2000, Sigma-Aldrich, USA) for 10 min. Finally, slides were coverslipped with Vectashield fluorescent mounting medium (Vector Laboratories, Burlingame, CA, USA). Appropriate controls were performed by omitting the primary antibodies to assure their specificity and to avoid autofluorescence. Fluorescence signals were analysed with a Nikon C2 confocal laser scanning microscope system (Nikon, Minato, Tokyo, Japan).
4
Quantifying Immune Cell Platforms
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Platforms were detected as described previously (Rotolo et al., 2005 (link)). Briefly, Jurkat cells were resuspended in RPMI 1640 supplemented with 1% heat-inactivated delipidated FBS, irradiated, and fixed in 2% PFA at appropriate times following exposure. Cells were blocked in 2% donkey serum in PBS on ice for 1 h, washed, and resuspended in 1:50 α-ceramide MID 15B4 (ALX-804-196-T050, Enzo Life Sciences) in PBS for 2 h. Cells were then washed with PBS + 0.1% Tween, resuspended in 1:300 CyTM3 AffiniPure goat anti-mouse IgG (H+L) in PBS + 0.1% Tween, and incubated for 1 h on ice. Cells were washed in PBS + 0.1% Tween and mounted on glass slides using Vectashield fluorescent mounting medium containing DAPI (Vector Laboratories). Platforms were imaged on a Leica SP5 Upright confocal microscope with HyD hybrid detector photon counter and HCX PL APO CS 40.0× 1.25 oil UV objective lens at room temperature. Acquisition was performed using Leica LAS AF software and counting of images with ImageJ v2.0 (National Institutes of Health). Platforms were quantified as percentage of cells exhibiting platform staining as indicated by condensed fluorescence to ∼25% of cell circumference.
5
Quantifying Muscle Vascular Density
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Immunohistochemistry was performed on 10-μm frozen sections of gastrocnemius muscle. After 5 minutes of fixation in 4% paraformaldehyde and rinsing in phosphate-buffered saline (PBS), immunostaining was performed as previously described.17 (link),29 (link) The slides were incubated overnight with vascular endothelial-cadherin (VE-cad; 1:200; CD144; BD Pharmingen; BD Biosciences, Franklin Lakes, NJ) rat anti-mouse primary antibody, followed by AlexaFluor 488 anti-rat secondary antibody, and washed and mounted in DAPI (4′,6-diamidino-2-phenylindole)-containing Vectashield fluorescent mounting medium (Vector Laboratories, Burlingame, CA). The sections were then scanned using an Axioscan microscope (Carl Zeiss, Oberkochen, Germany). The VE-cad–positive area of whole muscle was quantified blindly using Fiji software, version 1.53f51 (available at: http://fiji.sc/Fiji ). Quantifications are expressed as a percentage of the VE-cad–positive area to the total surface area of the gastrocnemius muscle.
6
Immunofluorescence Analysis of Intestinal Tissues
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Human colon biopsies and mice bowel samples were embedded into Shandon cryomatrix (Thermo Fisher Scientific) and cut into 5 μm slides, stored at -80 °C until use. HT-29 and CCD-18Co cells were seeded in chambers and cultured for 24 h in 37 °C. After repeated washing slides were permeabilized with Cytofix/Cytoperm (BD) for 15 min at RT, then incubated with primary antibodies specific to αSMA (sc-53015; mouse, 1:2000, Santa Cruz Biotechnology, Dallas, TX, USA) or IL-20RB (ab124332; rabbit, 1:100, Abcam) for 1 h at RT. After repeated washing slides were incubated with Alexa Fluor 488 goat anti-mouse IgG (A11001, Invitrogen) and Alexa Fluor 568 donkey anti-rabbit secondary antibody (A10042, Invitrogen), both diluted to 1:100 for 30 min at RT in the dark and counterstained with Hoechst 33,342 (1:2000, Sigma-Aldrich). Finally, slides were rinsed in PBS and coverslipped with Vectashield fluorescent mounting medium (Vector Laboratories, Burlingame, CA, USA). Appropriate controls were performed omitting the primary antibodies to assure their specificity and to avoid autofluorescence. Sections were analyzed with a Nikon C2 confocal laser scanning microscope system (Nikon, Minato, Tokyo, Japan).
7
Immunofluorescent Labeling of Axons
8
Retrograde Labeling of Retinal Ganglion Cells with FG
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RGC were retrogradely labelled with FG (Cambridge Bioscience, Cambridge, UK) by injecting 2 μL of a 4% FG solution into the optic nerve, proximal to the ONT site, 48 h before enucleation [34 (link),35 (link),42 (link),43 (link)]. Retinae (n = 16/group) were immersion-fixed for 2 h in 4% formaldehyde (TAAB Laboratories, Berkshire, UK), adhered onto Superfrost Plus microscope slides (VWR international, Leicestershire, UK) as whole mounts and cover slipped in Vectashield fluorescent mounting medium (Vector Laboratories, Peterborough, UK). Retinae were randomized and examined using an epifluorescent microscope (Zeiss, Hertfordshire, UK) and photographed with an AxioCam HRc digital camera controlled by Axiovision 4 software (all from Zeiss). FG+ RGC were counted using the automated function in ImagePro Software, version 6.0 (Media Cybernetics, Bethesda, USA) from images of 12 rectangular areas (0.36 × 0.24 mm), 3 from each quadrant of each retina, placed at standard radial distances from the centre of the optic disc at the inner (1/6 eccentricity), mid-periphery (1/2 eccentricity), and peripheral retina (5/6 eccentricity) and RGC densities computed as mean RGC density/mm2 (n = 8 rats/group, 16 retinae/group) [41 (link)].
9
Immunofluorescent Staining of Primary Microglia
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Primary adult microglial cells on glass cover slips fixed with 4% PFA were double-labelled for ANXA1 and pan-marker of microglia cell-type, Iba1. Cells were blocked with blocking buffer for 1 h at RT before incubation with rabbit anti-ANXA1 (1:200, Cell Signaling Technology, USA) and goat anti-Iba1 (1:200, Novus Biologicals, USA) overnight at 4 °C. Alexa Fluor® 568 anti-goat secondary antibodies and Alexa Fluor® 488 anti-rabbit secondary antibodies (1:500, Invitrogen, USA) were applied for 1 h at RT. DAPI DNA counterstain (1:5000) AbD Serotec, UK) was added to the cells for 10 min. Cells were washed with washing buffer before mounting with Vectashield Fluorescent Mounting Medium (Vector Laboratories, USA). The coverslip edges were sealed and stored in dark 4 °C until imaging with LSM 510 confocal (Carl Zeiss, Germany).
10
Choroidal Flatmount Imaging for L-CNV Analysis
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On day 14 post L-CNV induction, mice were euthanized by isoflurane overdose followed by cervical dislocation. The eyes were enucleated and fixed in 4% PFA in PBS for 1 h at 4 °C. The anterior portion including lens and the retina were removed, then the posterior eyecups were dissected out and underwent further fixation in 4% PFA in PBS overnight. The fixed eye cups were washed in blocking buffer (0.3% Triton X-100, 5% bovine serum albumin (BSA) in PBS) for 2 h at 4 °C. The eyecups were then stained for vasculature using Alexa Fluor 488 conjugated Griffonia simplicifolia isolectin B4 (GS-IB4; Molecular Probes, Thermo Fisher Scientific) at 1:250 dilution in buffer containing 0.3% Triton X-100, 0.5% BSA in PBS, overnight at 4 °C. The posterior eyecups were washed three times with PBS and mounted in Vectashield fluorescent mounting medium (Vector Laboratories) and coverslipped. Confocal imaging and analysis of L-CNV lesion volume were performed as previously described [64 (link),65 (link)]. Choroidal flatmount images were acquired using a Zeiss LSM 700 laser scanning confocal microscope with ZEN imaging software. The area measurements and volume calculations were performed using the freehand tool in ImageJ software.
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