5×105 bone marrow-derived macrophages were cultivated on 8-well tissue culture chambers (Sarstedt). After 7 days of differentiation and overnight activation, the M1 and M2 macrophages were incubated with 200 μM M2pep or scM2pep for 30 min at 37°C. The peptides were labeled to Streptavidin-PE-CF594 (BD Horizon). In order to achieve higher signal intensity, we used 200 μM M2pep in the immunofluorescence experiments, which did not alter the ability of M2pep to discriminate between M1 and M2 macrophages. The immunostaining was done as previously described [18 (link)]. Anti-mouse F4/80 (1:50; eBioscience) and Phalloidin-Alexa 488 (Invitrogen) were used as primary antibodies. The secondary antibody for F4/80 was Alexa-Flour 633 (Invitrogen). Nuclei were stained with Hoechst 33342 (Sigma-Aldrich). All coverslips were mounted on slides with Permount toluene solution (Fisher Chemicals) and imaged using an Olympus Fluoview FV1000 confocal microscope.
Permount toluene solution
Manufactured by Thermo Fisher Scientific
Permount toluene solution is a mounting medium used for preparing permanent microscope slides. It is a clear, viscous liquid that allows for the preservation and stabilization of specimen samples mounted on microscope slides.
Automatically generated - may contain errors
Lab products found in correlation
Nbt bcip, by Roche (2 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (2 mentions)
Blocking reagent, by Roche (2 mentions)
Anti dig alkaline phosphatase antibody, by Roche (2 mentions)
Alexa 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Fluoview fv1000 confocal microscope, by Olympus (1 mentions)
Streptavidin pe cf594, by BD (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Anti mouse f4 80, by Thermo Fisher Scientific (1 mentions)
8 well tissue culture chambers, by Sarstedt (1 mentions)
Mzfliii dissecting microscope, by Leica (1 mentions)
Fluoview 1000 confocal microscope, by Olympus (1 mentions)
Rat anti mouse cd68, by BioLegend (1 mentions)
Alexa fluor 546 conjugated goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Tax1bp1, by Abcam (1 mentions)
Bluing reagent, by Thermo Fisher Scientific (1 mentions)
Harris hematoxylin, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Mgcl2, by Thermo Fisher Scientific (1 mentions)
X gal, by Thermo Fisher Scientific (1 mentions)
10 protocols using permount toluene solution
1
Macrophage Polarization Analysis
2
Carmine Alum Staining of Mammary Glands
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Briefly, the inguinal mammary gland was spread onto a positively charged slide (Fisherbrand 12-550-15) and left in Clarke’s Fluid (75% ethyl alcohol, 25% acetic acid) overnight. The following day, glands were placed in 70% ethyl alcohol for 30 min before being stained in carmine alum (0.2% carmine, 0.5% potassium aluminium sulphate) overnight. Glands were destained for 4 to 6 h with destaining solution (1% HCl, 70% ethyl alcohol) and subsequently dehydrated in ascending concentrations of alcohol (15 min each 70, 95, 100% ethyl alcohol) before being cleared in xylene overnight. Slides were mounted with Permount toluene solution (Fisher Scientific) before imaging on a Leica MZFLIII dissecting microscope (University of Windsor). Images were captured using Northern Eclipse software.
3
Cst3 RNA In Situ Hybridization
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RNA in situ hybridization was performed as previously described (Touahri et al., 2015 (link)). Briefly, digoxygenin labeled RNA riboprobes for Cst3 (cDNA Image clone ID: 3593373) were made using T7 (Invitrogen, for anti-sense probe; Restriction Enzyme: SalI) and SP6 (Biolabs, for negative control sense probe; Restriction Enzyme: Not1) RNA polymerases and digRNA labeling mix following the manufacturer’s (Roche) protocol. RNA in situ hybridization was performed on 10μm sections collected on SuperFrost Plus slides (Thermo Scientific). Sections were hybridized with sense and anti-sense Cst3 probes and incubated overnight at 65°C. Sections were then blocked (2% blocking reagent; Roche, made up in 10% normal horse serum) for 60 mins at room temperature, incubated overnight with anti-dig alkaline phosphatase antibody (Roche), and then stained with an NTMT (100 mM NaCl, 50 mM MgCl, 100 mM Tris pH 9.5, Tween 20) - levamisole solution containing NBT-BCIP (Roche) substrate. Sections were mounted post-staining using Permount Toluene solution (Fisher Scientific).
4
Cst3 RNA In Situ Hybridization
5
Picrosirius Red Collagen Quantification
6
Immunofluorescence Imaging of Tax1BP1 and CD68 in Mouse Liver
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Tax1BP1+/+ mice were sacrificed and the liver tissue was fixed with 4% paraformaldehyde for 24 h. Ten micrometer-cryosections were prepared, rehydrated in PBS and blocked with 2% bovine serum albumin (BSA)/PBS solution containing 0.1% Triton X-100. The tissue was incubated with rabbit anti-TRAF6BP (= Tax1 BP1; 1:100 dilution; Abcam) and with rat anti-mouse CD68 (1:100 dilution; Biolegend) in PBS containing 2% BSA and 0.1% Triton X-100 overnight at 4 °C. After washing with PBS, the sections were further incubated with Alexa Fluor 546 conjugated goat anti-rabbit (1:200 dilution; Invitrogen) and Alexa Fluor 633 conjugated goat anti-rat (1:200 dilution; Invitrogen) for 1 h at room temperature. Both secondary antibodies were diluted in PBS containing 2% BSA and 0.1% Triton X-100. Sections without rabbit anti-TRAF6BP staining were used as negative controls. All coverslips were mounted on slides with Permount toluene solution (Fisher Chemicals) and imaged using an Olympus Fluoview 1000 confocal microscope.
7
Hematoxylin and Eosin Staining of Liver Tissue
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Sections of fresh frozen liver tissue were sectioned at 8 μm and dehydrated in absolute methanol for 1 min. The slides were stained in Harris’ Hematoxylin (Sigma Aldrich, St. Louis, MO) for 2 min, followed by a 20 second wash in water. The pH was then reduced with a 1 minute wash in bluing reagent (Fisher Scientific, Cambridge, MA). The slides were washed for 30 seconds in water, followed by 30 seconds in 95% ethanol, and then stained in Eosin Yellow (Fisher Scientific) for 1 minute. The sections were then dehydrated in increasing concentrations of ethanol, followed by xylenes. Finally, the slides were mounted in Permount toluene solution (Fisher Scientific) and imaged using light microscopy.
8
Mammary Gland LacZ Staining Protocol
9
Quantifying Collagen in Duct Epithelia
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The sections were deparaffinized in xylene and rehydrated in water. Masson’s trichrome staining kit (DAKO, Cat#AR173) was performed following the manufacturer’s procedure protocol and mounted using Permount toluene solution (Thermo Fisher Scientific). Triplicate images of the stained sections were collected for representative areas of 2560 × 1920 pixels (0.44 μm/pxls) using a Zeiss AX10 light microscope. The positive blue stain was converted to black using Adobe Photoshop CS5’s (version 12.0 × 64) magic wand tool. The images were imported into ImageJ (version 1.46r), and the black selections were quantified as percent area positive for collagen. The images were normalized against the periphery of the duct epithelia in which the final ratio represents collagen/epithelia.
10
Cell Invasion Assay Using Matrigel
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Cell permeable membrane inserts (Falcon, #08-771-21) were coated with 200 μL of diluted 1:100 growth factor-reduced Matrigel basement membrane matrix (Corning, #356230) in cold PBS. Inserts were placed in 24-well plates (Falcon), and artificial extracellular matrix was allowed to polymerize for 2 h at room temperature. Following polymerization incubation, excess Matrigel solution was removed by pipette. Fifty thousand cells in 500 μL of serum-free DMEM were seeded atop the polymerized Matrigel in the inserts. Five hundred microliters of complete DMEM containing 10% FBS was added to each lower chamber. Invasion assays were incubated at 37 °C/5% CO2. After 48 h, invaded cells were fixed and stained using the Hema 3 Manual Staining Stat Pack (Thermo Fisher Scientific, #23-123-869) according to the manufacturer’s protocol and mounted using Permount toluene solution (Thermo Fisher Scientific). All experiments were done in technical triplicates.
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