Mouse tissues were fixed by transcardial perfusion with 4% paraformaldehyde in cold PBS. The sacral DRG (S1–S4) were excised and prepared for frozen sectioning. DRG were sectioned at 15µm. Sections were placed in blocking solution for 1 hour at room temperature. Then sections were incubated with either rabbit anti-p-ERK1/2 (1:50; Cell Signaling), rabbit anti-PAR2 (1:50; Santa Cruz; sc-13504), or anti-goat isotype (1:50; Santa Cruz; sc-2028) at 4°C overnight. The sections were washed 3 times in PBS for 5 min and placed in Alexa 488 anti-rabbit secondary antibody (1:400; Invitrogen; A11034) for 1–2hrs at room temperature in the dark. Sections were washed 3 times 5 min each in PBS. Slides were covered with mounting medium containing DAPI (Invitrogen; 1319493) and sealed with a coverslip (Corning). Immunohistochemical tissues were sectioned and processed at the Northwestern pathology core. Briefly, tissue were fixed in 10% formalin and embedded in paraffin and stained for either toluidine blue or hematoxylin & eosin (H&E). Images were viewed with a Leica DMI 6000B inverted microscope and z stacks were deconvolved using AutoQuant Deconvolution algorithms by Media Cybernetics.
Coverslip
Manufactured by Corning
Sourced in United States
Corning Coverslips are thin, optically clear glass or plastic sheets used to cover and protect samples in microscopy and other laboratory applications. They serve the core function of providing a flat, transparent surface to cover and protect samples, enabling clear visualization and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 700, by Zeiss (2 mentions)
Dmi6000b inverted microscope, by Leica (1 mentions)
Sc 2028, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti p erk1 2, by Cell Signaling Technology (1 mentions)
Rabbit anti par2, by Santa Cruz Biotechnology (1 mentions)
Glass slide, by Corning (1 mentions)
Spinsr10 microscope, by Olympus (1 mentions)
Six well plate, by Corning (1 mentions)
Lab tek 2, by Thermo Fisher Scientific (1 mentions)
Ix71 inverted microscope, by Olympus (1 mentions)
Pab1620, by Abcam (1 mentions)
Pab240, by Abcam (1 mentions)
Alexa fluor 488 goat anti mouse igg secondary antibody, by Thermo Fisher Scientific (1 mentions)
5 protocols using coverslip
1
Immunohistochemical Analysis of DRG Neurons
2
Visualizing Lipid Distribution in Arabidopsis
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One-week-old Arabidopsis thaliana, WT (Col), and pldα1/δ knockout mutants were incubated with Nitrobenzoxadiazole (NBD) labeled phospholipids; NBD-PC or NBD-PA on a Corning glass slide (cat#2948) and covered with Corning coverslip (cat# 2980). Images were taken on an Olympus SpinSR10 microscope using a 40x/1.4 oil objective lens. Photos were taken in a z-stack of 30 µm thickness with a z-step size of 1µm. Fluorescence was detected using a 464 nm excitation and 531 nm emission. Obtained images were then projected on the z-axis using ImageJ (Fiji) software.
3
Mouse Leydig and Adrenocortical Cell Culture
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The MA-10 mouse Leydig tumor cell line (a gift of Dr. Mario Ascoli) was derived from the Leydig tumor, M5480P (28 (link)). The Y-1 cell line (a gift of Dr. Bernard Schimmer) was derived from a mouse adrenocortical tumor (29 (link)). General procedures for cell culture were as described previously (1 (link)). A day before the experiment, approximately 3–6 × 105 cells were seeded onto the coverslip (Corning) coated with ploy-Lysine (Sigma) in wells of a six-well plate (Corning).
4
Visualization of ZIKV E Protein Localization
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To confirm the ZIKV E protein presence at the cell membrane surface level, an IF assay was performed as follows: The mosquito C6/36 cells were seeded on an 8-well separation chamber slide system (Lab-Tek II; Thermo Fisher Scientific), incubated until confluence, and infected as described above. The cells were fixed with 2% paraformaldehyde for 5 min at 4 °C, blocked for nonspecific binding sites with 2% BSA for 30 min at RT, and washed with 0.5% BSA. The cells were stained with mouse anti-ZIKV E protein antibody at a 1:300 dilution in 0.5% BSA and incubated overnight at 4 °C with constant 1000 rpm agitation. After washing with 0.5% BSA, the Alexa Fluor 555-conjugated anti-mouse IgG was added at a 1:500 dilution in 0.5% BSA, incubated for 2 h at RT, and washed with 0.5% BSA. The separation chamber was then withdrawn, the slide was covered with mounting medium with DAPI (FluoroQuest; AAT Bioquest, Sunnyvale, CA, USA), and a coverslip (Corning) was placed. The samples were observed via fluorescence microscopy (Olympus IX71 inverted microscope; Olympus Corp.), and the images were analyzed with ImageJ software.
5
Immunofluorescence Detection of p53 Status
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The cells were seeded on a coverslip (Corning Life Sciences, United States) and then incubated at 37°C overnight. Subsequently the cells were fixed with 4% paraformaldehyde for 10 min at room temperature. The cell membrane was permealized with 0.5% Triton X-100 for 5 min to allow the entry of antibody into cells. Then the permealized cells were blocked with 1% BSA (bovine serum albumin) for 1 h at room temperature, followed by incubation with the primary antibody PAB1620 or PAB240 (Abcam, United States) at 4°C overnight. These antibodies can recognize the p53 protein status. PAB1620 and PAB240 are able to specifically recognize wild-type and mutated p53, respectively (Yu et al., 2012 (link)). Thereafter, the cells were incubated with the goat anti-mouse IgG Alexa Fluor 488 secondary antibody (Invitrogen, United States) for 1 h at room temperature. The nuclei were stained with DAPI (4′, 6-diamidino-2- phenylindole) for 10 min at room temperature. After washes with PBS, the immunofluorescence of cells was detected by laser scanning confocal microscope LSM700 (Zeiss, Germany).
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