Isolated neutrophils were fixed in 4% (vol/vol) paraformaldehyde (PFA) (biosharp, China), blocked with 3% (wt/vol) bovine serum albumin (BSA) (Solarbio, China) for 30min at 37°C and then incubated with the primary antibody Histone H3 (citrulline R2+R8+R17, ab5103, 1:300, abcam, USA), Alexa Fluor 647 conjugated secondary antibody (1:1000, Invitrogen, USA) at 1:300 dilution and FITC anti-myeloperoxidase antibody (ab11729, 1:10, Abcam) according to a previously described protocol (21 (link)). Flow cytometric analysis was operated using an Beckman CytoFLEX flow cytometer.
Paraformaldehyde pfa
Manufactured by Biosharp
Sourced in China
Paraformaldehyde (PFA) is a solid polymer of formaldehyde used as a fixative in various laboratory applications. It is commonly employed in histology, cell biology, and microscopy to preserve and stabilize biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 647 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Solarbio (1 mentions)
Ab11729, by Abcam (1 mentions)
Cytoflex flow cytometer, by Beckman Coulter (1 mentions)
Ab5103, by Abcam (1 mentions)
Imager z2, by Zeiss (1 mentions)
Eclipse ts2, by Nikon (1 mentions)
Triton x 100, by Beyotime (1 mentions)
Fish kit, by GenePharma (1 mentions)
Fluorescein isothiocyanate (fitc), by Boster Bio (1 mentions)
Apotome 2, by Zeiss (1 mentions)
Pkh67, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by GeneTex (1 mentions)
Alexa fluor 594 conjugated goat anti rabbit igg h l, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated goat anti mouse igg h l, by Thermo Fisher Scientific (1 mentions)
Optimal cutting temperature compound o c t, by Sakura Finetek (1 mentions)
Dapi c0060, by Solarbio (1 mentions)
6 protocols using paraformaldehyde pfa
1
Neutrophil Immunophenotyping by Flow Cytometry
2
Identification of M0 Macrophages from THP-1 Cells
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THP-1 cells in the subculture in the logarithmic growth period were inoculated in 12-well glass bottom plates at 5 × 105 cells/well and induced under the optimal concentration and action time of PMA. M0 cells were identified by observing the cell morphology and immunofluorescence staining. The cell morphology was observed using a light microscope (E-CLIPSE Ts2; Nikon, Japan). For immunofluorescence staining, the cells were fixed with 4% paraformaldehyde (PFA) (Biosharp, Anhui, China) and incubated with diluted rabbit primary antibody against CD68 (1:300) (Boster, Wuhan, China) overnight at 4 °C. The sections were incubated with a secondary antibody (rabbit, 1:300; Absin, Shanghai, China) in the dark at room temperature for 1 h on the following day. Antifade Mounting Medium with DAPI (Servicebio, Wuhan, China). Slides were analyzed under a fluorescence microscope (Zeiss Imager Z2; Zeiss, Germany).
3
Localization of BMPR1B-AS1 in Ishikawa Cells
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Digoxigenin (DIG)-labeled BMPR1B-AS1 probes were designed and synthesized by BOSTER (Wuhan, China). Fluorescent in situ hybridization experiments were conducted using a FISH Kit (GenePharma) according to the manufacturer’s instructions. Briefly, Ishikawa cells were fixed in 4% paraformaldehyde (PFA, Biosharp, Hefei, China) at room temperature for 15 min and then washed with PBS. The cells were then permeabilized with Triton X-100 (Beyotime, Shanghai, China) at 4°C for 15 min. Subsequently, the cells were incubated with DIG-labeled BMPR1B-AS1 probes or Control-FISH probes at 55°C for 4 h and washed with 2× PBS for 10 min. The signals were detected with SABC (streptavidin-biotin complex)-FITC (BOSTER). DAPI was used to counterstain the nuclei. Images were obtained by confocal microscopy. The probe sequences are presented in Table 1 .
4
Labeling and Tracking Exosome Uptake in Cells
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MG-Exos were labeled with a red fluorescent lipophilic dye PKH67 (Sigma) to monitor the motion of the exosomes. MG-Exos were resuspended in sterile PBS and incubated with 5 μM PKH67 for 15 min. Then, the suspension containing labeled exosomes was washed twice at 140,000×g for 90 min and resuspended in sterile PBS and used for the uptake experiment.
The bEnd.3 cells were seeded on chambered culture slides in 24-well cell culture plates until reaching 80% confluence and then incubated in the completed medium containing 200 μg/ml PKH67-labeled exosomes at 37°C for 12 h. Culture slides were fixed with 4% paraformaldehyde (PFA; Biosharp, China) for 20 min and then incubated with 5% BSA (Biofroxx, Germany) in PBS for 30 min to block nonspecific staining. Nuclei were stained with DAPI (GeneTex Inc., USA), and signals were analyzed with a fluorescence microscope (Zeiss Apotome 2, Zeiss, Germany).
The bEnd.3 cells were seeded on chambered culture slides in 24-well cell culture plates until reaching 80% confluence and then incubated in the completed medium containing 200 μg/ml PKH67-labeled exosomes at 37°C for 12 h. Culture slides were fixed with 4% paraformaldehyde (PFA; Biosharp, China) for 20 min and then incubated with 5% BSA (Biofroxx, Germany) in PBS for 30 min to block nonspecific staining. Nuclei were stained with DAPI (GeneTex Inc., USA), and signals were analyzed with a fluorescence microscope (Zeiss Apotome 2, Zeiss, Germany).
5
Immunohistochemical Staining of Brain Tissue
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Nissl staining solution (G1036) was purchased from Servicebio (Wuhan, China). Corn oil was purchased from Maclin Biochemical (Shanghai, China). CCT007093 was purchased from MedChemExpress (New Jersey, USA). Resveratrol was purchased from Aladdin (Shanghai, China). Optimal cutting temperature compound (OCT) was purchased from Sakura Finetek (Torrance, USA). Paraformaldehyde (PFA) was purchased from Biosharp (Hefei, China). Antibodies: Rabbit anti-Iba1 (ab178846) and Rabbit anti RBPMS (ab152101) were purchased from Abcam (Cambridge, UK). Mouse anti IL-1β (#12242) were purchased from Cell Signaling Technology (Boston, USA). Alexa Fluor 594–conjugated goat anti-rabbit IgG (H+L) and Alexa Fluor 488–conjugated goat anti-mouse IgG (H+L) were purchased from Thermo Fisher Scientific (Waltham, USA).
6
Cellular Uptake of CY5-Labeled Compounds
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PANC1 cells (1 × 105 cells/well) were seeded into the confocal imaging chambers. After 24 h, the cells were treated with CY5-DM, CY5-DM@SBE-β-CD, and CY5-DM@SAC5A (10/10 μM). After a 6 h incubation under normoxic conditions, the culture medium was exchanged with fresh medium. Thereafter, the cells were incubated at 37 °C for 18 h under either normoxic or hypoxic conditions, respectively. Subsequently, the cells were fixed in 4% paraformaldehyde (PFA; Biosharp, Hefei, China) for 15 min, washed with PBS buffer three times and then imaged using CLSM. Cell nuclei were counterstained with DAPI (C0060, Solarbio, Beijing, China) for 5 min.
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