ChAT-IRES-Cre × Ai32 transgenic mice (N=4) were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg). A surgical plane of anesthesia was maintained throughout the procedure with supplements of ketamine (50 mg/kg) as needed. We then injected 0.3 μl of red retrobeads (LumaFluor Inc.) into A1 at 0.05 – 0.1 μl/min using a motorized injector (Stoelting Co.). After allowing 7 days for retrograde transport, mice were deeply anesthetized with ketamine and prepared for transcardial perfusion with a 4% formalin solution in 0.1M phosphate buffer. The brains were extracted and post-fixed at room temperature for an additional 12 hours before transfer to 30% sucrose solution. Coronal sections (30 μm thick) were prepared on a cryostat and counterstained with DAPI (VECTASHIELD HardSet Antifade Mounting Medium with DAPI). The location of the injection site in ACtx was confirmed in each case (Fig. S4 ). All sections containing the basal forebrain were analyzed by counting the number of bead+ and ChAT+ cells. Bead+ cells were defined as distinct oval shapes with more than 10 bead pixels inside and a DAPI-stained nucleus in the middle. ChAT+ cells were identified based on the shape and the intensity of the EYFP signal.
Hardset antifade mounting medium with dapi
Manufactured by Vector Laboratories
Sourced in United States
HardSet Antifade Mounting Medium with DAPI is a solution used to mount and preserve fluorescently labeled samples for microscopic analysis. It contains an antifade agent to protect against photobleaching and DAPI, a fluorescent dye that binds to DNA, allowing for the visualization of cell nuclei.
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Lab products found in correlation
Falcon 8 well culture slide, by BD (2 mentions)
Blocking one histo, by Nacalai Tesque (2 mentions)
Motorized injector, by Stoelting (1 mentions)
Trueview autofluorescence quenching kit, by Vector Laboratories (1 mentions)
Alexa fluor 488 conjugated anti rabbit igg, by Abcam (1 mentions)
Bz x700 fluorescence microscope, by Keyence (1 mentions)
Axiovision z1, by Zeiss (1 mentions)
Alexa fluor 488 anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Dmi4000b, by Leica (1 mentions)
Bz x710 fluorescence microscope, by Keyence (1 mentions)
Citrate buffer, by Abcam (1 mentions)
Anti lc3a b antibody, by Cell Signaling Technology (1 mentions)
7 protocols using hardset antifade mounting medium with dapi
1
Retrograde Tracing of Cholinergic Neurons
2
Immunofluorescence Staining of Formalin-Fixed Paraffin-Embedded Tumor Tissue
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The formalin-fixed tumor tissues were embedded in paraffin, and 4-μm thick sections were obtained. The sections were stained with the primary antibodies—purified anti-mouse CD3 (CD3-12, rat IgG1) and anti-mouse Melan-A (EPR20380, rabbit IgG)—and the secondary antibodies—Alexa Fluor 488-conjugated anti-rabbit IgG and Alexa Fluor 594-conjugated anti-rat IgG. The antibodies were purchased from Abcam. The stained sections were treated with the TrueVIEWTM autofluorescence quenching kit (VECTOR Laboratories, Burlingame, CA, USA), mounted with HardSet Antifade Mounting Medium with DAPI (VECTOR Laboratories), and analyzed using a BZ-X700 fluorescence microscope (KEYENCE, Osaka, Japan).
3
Autophagy Imaging and Corneal Epithelial Turnover
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Eyes of GFP-LC3 transgenic mice, which systemically express GFP fused to LC3 (Mizushima et al., 2004 (link)), were rapidly dissected out and embedded in optimal cutting temperature compound rapidly. 5-µm frozen sections were fixed in 4% PFA at RT for 10 min. After three washes with PBS, the sections were mounted using HardSet Antifade Mounting Medium with DAPI (VectaShield). Images were taken using a 100× objective in an epifluorescence microscope AxioVision Z1 (ZEISS). DAPI was used to counterstain nuclei, and GFP punctas were counted. The Beclin-1+/− mouse has been described (Qu et al., 2003 (link)). Central corneal epithelia of Beclin-1+/− and Beclin-1+/+ mice were removed by application of a rotating diamond burr to the surface of the central cornea. The limbal epithelium remained intact. Tissues were embedded in paraffin blocks for immunohistochemical analysis of BrdU. Animal procedures were approved by the Northwestern University Animal Care and Use Committee.
4
Identification of MDSC Subsets in Tumor-Bearing Mice
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In tumor‐bearing mice, MDSC consisted of two major subsets, namely Ly6G−Ly6Chigh monocytic MDSC and Ly6G+Ly6Clow granulocytic MDSC.26 Dual immunofluorescence staining was done with antibodies specific to Ly‐6c and Ly‐6 g (Table S1 ) for resected tumors. Tumors were embedded immediately in Tissue‐Tek OCT Compound (Sakura Fine Technical, Tokyo, Japan) to generate 6‐μm‐thick cryosections on glass slides. Sections were fixed in methanol at 4°C for 10 minutes, which was followed by blocking and permeabilization in 1% BSA/0.3% Triton X‐100 for 1 hour. The sections were then incubated with anti‐Ly‐6c and Ly‐6g antibodies (dilution 1:50 for both) at 4°C overnight. Next, the sections were incubated with Alexa Fluor 488 anti‐mouse IgG and Alexa Fluor 568 anti‐rat IgG secondary antibodies (dilution 1:1000; Life Technologies) for 60 minutes, rinsed three times in PBS, and mounted with HardSet antifade mounting medium with DAPI (Vector Laboratories). The sections were examined under a fluorescence microscope (Leica DMI 4000B).
5
Immunofluorescence Assay for LC3 Autophagy Marker
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Cells were cultured on Falcon 8‐well Culture Slides (354118, BD Falcon, Corning, Inc.), then fixed with 4% paraformaldehyde for 20 min at room temperature, permeabilized with 0.1% Triton X‐100, washed with PBS, and blocked with Blocking One Histo (Nacalai Tesque). Then, they were incubated with anti‐LC3A/B antibody (1:100) overnight at 4℃ then with Alexa Fluor 488‐conjugated goat anti‐rabbit IgG (H+L) cross‐absorbed secondary antibody (A‐11008, 1:200, Life Technologies, Tokyo, Japan) for 2 h at with shading. Nuclei were stained with HardSet Antifade Mounting Medium with DAPI (H‐1500, Vector Laboratories) for 15 min. Slides were imaged with a KEYENCE BZ‐X710 fluorescence microscope (Keyence Corp.).
6
Immunofluorescence Analysis of Transcription Factors and MET Expression
7
Immunofluorescence Assay of LC3A/B
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The cells were cultured on Falcon 8-well Culture Slides (354118, BD Falcon, Corning, Inc., Corning, NY, USA), then xed with 4% paraformaldehyde for 20 min at room temperature, permeabilized with 0.1% Triton X-100, washed with PBS, and blocked with Blocking One Histo (Nacalai Tesque, Kyoto, Japan). Then, they were incubated with anti-LC3A/B antibody (1:100, Cell Signaling Technology) overnight at 4°C, and goat anti-rabbit IgG (H + L) cross-absorbed secondary antibody, Alexa Flour 488 (A-11008, 1:200, Life Technologies, Tokyo, Japan) for 2 h at room temperature (20-25°C) with shading. Nuclei were stained with HardSet Antifade Mounting Medium with DAPI (H-1500, Vector Laboratories, CA, USA) for 15 min. Finally, we observed the slides using a uorescence microscope system, KEYENCE BZ-X710 instrument (Keyence Corp., Osaka, Japan).
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