At sacrifice, mouse heart samples were frozen by immersion in isopentane cooled in liquid nitrogen. For immunohistochemistry analysis, heart samples were mounted in optimal cutting temperature compound (OCT) and sectioned on a cryostat at −20 °C to a thickness of 8 μm. Sections were fixed in 10% neutral buffered formalin (SIGMA, HT501128) at RT. After three washes in PBS, samples were permeabilized using 0.2% Triton X- 100 in PBS for 15 min at RT, followed by three washes in PBS, and then blocked in 5% normal goat serum + 2% BSA in PBS for 1 h at RT. After three washes in PBS, samples were incubated with the rabbit anti-HA-Tag (Cell Signaling, 3724) primary antibody 1:2000 in DAKO solution (Agilent, S3022) for 1 h at RT, followed by three additional washes in PBS. Sections were then incubated with goat anti-rabbit IgG (H + L) AlexaFluor diluted 1:300 in DAKO diluent solution 568 for 1 h at RT, washes three times in PBS and finally coverslip with prolonged diamond antifade mountant with DAPI (Thermo Fisher, P36962). All confocal images were acquired using a Zeiss LSM880 microscope using identical acquisition parameters.
P36962
Manufactured by Thermo Fisher Scientific
Sourced in United States
The P36962 is a laboratory equipment product from Thermo Fisher Scientific. It is a specialized instrument designed for scientific applications. The core function of this product is to perform a specific set of laboratory tasks, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 880 confocal microscope, by Zeiss (4 mentions)
Adi 905 173 100, by Enzo Life Sciences (3 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (3 mentions)
G9023, by Merck Group (3 mentions)
Axio observer z1, by Zeiss (3 mentions)
Model bz x710, by Keyence (2 mentions)
Vectashield mounting medium, by Vector Laboratories (2 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Prolong diamond antifade mountant, by Thermo Fisher Scientific (2 mentions)
A11035, by Thermo Fisher Scientific (2 mentions)
A11029, by Thermo Fisher Scientific (2 mentions)
Leica tcs sp5 confocal microscope, by Leica camera (2 mentions)
Goat serum, by Merck Group (2 mentions)
Ab92547, by Abcam (2 mentions)
Sp5 3 confocal microscope, by Leica camera (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Vs120 microscope, by Olympus (2 mentions)
Cryostat, by Leica camera (2 mentions)
38 protocols using p36962
1
Immunohistochemical Analysis of Mouse Hearts
2
Confocal Microscopy Analysis of Cells
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Confocal microscopy was performed as suggested elsewhere (Cheng et al., 2018 (link)). Briefly, the cells were fixed in 4% paraformaldehyde at the room temperature for 10 min. Then, the cells were blocked with 0.5% BSA for 30 min at the room temperature, followed by incubation with the antibodies CD31 (1:100; #3528; Cell Signaling) and Oct4 (1:200; #2750; Cell Signaling) for overnight at 4°C. Finally, incubated with anti-mouse and anti-rabbit IgG, respectively, conjugated with Alexa Flour 488 (1:1000; A-10680; Thermo) and Alexa Flour 647 (1:1000; A-32795; Thermo) for 50 min at the room temperature. Subsequently, the coverslips were treated with DAPI (P36962; Thermo) and visualized under a laser scanning confocal microscope (A1+; Nikon).
3
Multimodal Imaging of Cultured Cells
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For brightfield and epifluorescence, cultured cells were imaged by REVOLVE4 microscope (ECHO) with a 10× objective. For confocal microscopy, samples in eight-well chamber slides were fixed in 4% paraformaldehyde for 10 min at room temperature and stained as previously described (74 (link)). Cells were permeabilized and stained with antibodies against DAPI (P36962, Thermo Fisher), LAMP1 (9091S, Cell Signaling), LBPA (MABT837, Sigma), and Rab4 (ab13252, Abcam) at room temperature for 1 h. For filipin staining, cells were fixed and incubated with 50 μg/mL filipin (SAE0087, Sigma-Aldrich) at 37 °C for 30 min. Stained cells were washed with PBS, whole-mounted with Antifade Mountant, and imaged with a Zeiss LSM880 Confocal Microscope at the Molecular Microbiology imaging core facility at Washington University in St. Louis. Images were visualized by Volocity v6.3 and quantification was determined by ImageJ (NIH).
4
Cross-linking and Imaging of ASC Speck Formation
5
Gamma-H2AX Immunostaining of IR-Treated Cells
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RT112 and stable cell lines were plated onto 8-well chamber slides (734–2050; NUNC, Roskilde, Denmark) and fixed with 100% methanol for 5 min at –20°C, at each time point after IR. Then cells were washed three times for 5 min each time in PBS. All subsequent steps were carried out at room temperature. Samples were blocked for 30 min in blocking buffer (1% BSA, 22.52 mg/ml glycine in PBST (PBS + 0.1% Tween 20)). Rabbit anti-phospho-histone H2A.X (Ser139) (#2577; Cell Signalling Technology, Danvers, MA, USA) was diluted 1:500 in PBST and incubated overnight at 4°C. Cells were washed with PBS, after which secondary antibody [AlexaFluor 568 (A11036; Thermo Fisher Scientific, Waltham, MA, USA)] was diluted 1:2000 in PBST and applied for 1 hr in room temperature. Cells were washed with PBS as before and mounted by using proLong diamond antifade mountant with DAPI (P36962; Thermo Fisher Scientific, Waltham, MA, USA). Cells were visualised for γ-H2AX immunostaining through confocal microscopy (Zeiss 710).
6
Immunohistochemical Staining of Salmonella and GFP
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Excised tumor sections were fixed in 10% formalin, embedded in paraffin and cut into 5 µm sections. Antigen retrieval was performed by incubating deparaffinized sections in 20 mM sodium citrate (pH 7.6) buffer for 20 min at 95 °C. Samples were rehydrated with DI water and Tris buffered saline with 0.1% Tween 20 (TBS-T). Prior to staining, tissue sections were blocked with Dako blocking buffer (Dako, catalog # X0909). Tissue sections were stained to identify Salmonella and GFP with (1) FITC-conjugated rabbit anti-Salmonella polyclonal antibody (Abcam, catalog # ab69253; 1:100 dilution), and (2) either rat anti-myc monoclonal antibody (Chromotek, catalog # 9e1-100; 1:100 dilution) or rat anti-GFP monoclonal antibody (Chromotek, catalog # 3h9-100; 1:100 dilution). Sections were incubated with Alexaflor-568 goat anti-rat secondary antibodies (ThermoFisher, catalog # A11077) and counterstained with DAPI-containing mountant (ThermoFisher, catalog # P36962).
7
Immunofluorescence Staining of Paraffin Sections
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Immunofluorescence paraffin sections were first deparaffinized and rehydrated, followed by high-pressure repair with sodium citrate buffer. Then, the sections were blocked with blocking buffer for 1 h (10% donkey serum in PBS+ 0.1% Triton X-100), incubated with primary antibody (4 °C, overnight), and incubated with secondary antibody (1 h), in sequence. After washing with PBS, the slides were mounted with the DAPI-containing mounting medium (Thermo, P36962, Waltham, MA, USA).
8
Embryonic Brain Development Labeling
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The specific experimental steps were carried out according to the EDU labeling kit manual (Beyotime, C0078S, Shanghai, China). Briefly, the female mice that detected the vaginal plugs were recorded as E0.5. At E12, the pregnant mice were intraperitoneally injected with EdU working solution (50 mg/kg), for two consecutive days for a total of 4 times, and the brains of embryonic mice were taken at E14 and fixed in paraformaldehyde. Paraffin sections were made according to routine procedures and immunofluorescence staining was performed, and then the prepared Click reaction solution was added dropwise to the tissue and incubated for 30 min. After washing, the slices were mounted with DAPI-containing mounting medium (Thermo, P36962, Waltham, MA, USA).
9
Visualization and Quantification of ASC Specks in Macrophages
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The WT alveolar macrophages and BMDMs were seeded on chamber slides. After LPS and poly(dA:dT) stimulation, cells were fixed with 4% paraformaldehyde and then incubated with polyclonal ASC antibody (ADI-905-173-100, Enzo Lifesciences) for 16 h and FITC goat anti-rabbit (IgG) secondary antibody (ab6717, Abcam (Cambridge, MA, USA)) for 1h followed by DAPI (P36962, ThermoFisher Scientific) staining [41 (link),42 (link),43 (link)]. The ASC specks were analyzed using a Zeiss LSM880 laser scanning confocal microscope and quantified using ImageJ software v1.52a (Bethesda, MD, USA). The graph in figure represents the quantification of percent of ASC speck-positive cells for each mouse.
10
Quantifying POMC Cells from Ucp2PomcKO Mice
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To validate POMC cells percentage in cell derived from Ucp2PomcKO and control mice, cells were treated with tamoxifen (to label POMC neurons with tdTomato expression) as a control group. After 48 h, cells were washed with pre-warmed 1x PBS and then incubated in 1 μM DAPI (Diluted in pre-warmed 1x PBS, Cat# P36962, Thermo Fisher Scientific) at RT for 5 min. After several washes with 1x PBS, coverslips were mounted on glass slides with a drop of Vectashield mounting medium (Cat# H-1000, Vector Laboratories) and five coverslips per group were then analyzed by capturing five random fields per coverslip observed cells on 20 x magnification by Fluorescence Microscope (Model BZ-X710, KEYENCE). For the quantitative analysis of cell numbers, tdTomato expressing cells in DAPI-stained were manually counted using ImageJ software.
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