An original method was applied to label proteins in the glioma neurospheres. Neurospheres were placed into cell insert (Millipore, US) and fixed by 0.4% Paraformaldehyde solution (Solarbio, China). Then, the neurospheres were washed three times in PBS, and incubated with primary antibodies (See Supplementary Table 1 ) overnight at 4°C. Alexa-Fluor 488 conjugate anti-rabbit secondary antibody (1:5000, Cell signaling, US) and Alexa-Fluor 594 conjugate anti-mouse secondary antibody (1:5000, Life technologies, US) were used for fluorescent double-staining. Serum medium cultured CD133+ cells were stained through Alexa-Fluor 594 conjugate phalloidin (1:200, Life technologies, US) for 20 minutes at 37°C to exhibit actin cytoskeleton. DAPI solution (Solarbio, China) was employed to label cell nucleus. Images were observed and captured by Perkinelmer UltraVIEW VOX confocal microscope (Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China).
Paraformaldehyde solution
Manufactured by Solarbio
Sourced in China
4% paraformaldehyde solution is a fixative used in various biological and histological applications. It is a concentrated aqueous solution of paraformaldehyde, a polymer of formaldehyde. This solution is typically used to preserve and stabilize biological samples, such as cells and tissues, prior to further processing and analysis.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Beyotime (3 mentions)
Dapi solution, by Solarbio (2 mentions)
Cell insert, by Merck Group (2 mentions)
Alexa fluor 594 conjugate anti mouse secondary antibody, by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Solarbio (2 mentions)
Triton x 100, by Solarbio (2 mentions)
Alexa fluor 488 conjugate anti rabbit secondary antibody, by Cell Signaling Technology (1 mentions)
Ultraview vox confocal microscope, by PerkinElmer (1 mentions)
Alexa fluor 488 conjugated anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions)
Fluorescein diacetate, by Merck Group (1 mentions)
Chloral hydrate, by Merck Group (1 mentions)
Milli q system, by Merck Group (1 mentions)
Chitosan, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Corning (1 mentions)
Cck 8 kit, by Beyotime (1 mentions)
Antifade, by Thermo Fisher Scientific (1 mentions)
Upper transwell chamber, by Thermo Fisher Scientific (1 mentions)
Crystal violet, by Beyotime (1 mentions)
Ckx53, by Olympus (1 mentions)
Masson trichrome, by Solarbio (1 mentions)
33 protocols using paraformaldehyde solution
1
Glioma Neurosphere Protein Labeling
2
Protein Labeling in Glioma Tumorspheres
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An original method was applied to label proteins in the glioma tumorspheres. Tumorspheres were placed into a cell insert (Millipore, US) and fixed with 0.4% paraformaldehyde solution (Solarbio, China). Then, the tumorspheres were washed three times with PBS, and incubated with primary antibodies overnight at 4 °C. Alexa-Fluor 488 conjugated anti-rabbit secondary antibody (1:1000, Life Technologies, USA) and Alexa-Fluor 594 conjugate anti-mouse secondary antibody (1,1000, Life Technologies, USA) were used for fluorescent double-staining. DAPI solution (Solarbio, China) was employed to label cell nuclei.
3
Pediatric Hydronephrosis and UPJO
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Clinical data of pediatric hydronephrosis caused by congenital UPJO were collected from the Affiliated Hospital of Zunyi Medical University from 2006 to 2012. B-mode ultrasound image, intravenous pyelography, and retrograde pyelography confirmed that UPJO resulted in hydronephrosis. The patients (46 males and 34 females) ranged in age from 3 days to 14 years, excluding cases with infectious hydronephrosis and pyonephrosis. All patients underwent open dismembered pyeloplasty and a small wedge of tissue from the narrow segment of the ureteropelvic junction was taken. According to the age, all cases were divided into newborns (aged 3 days–1 year, 20 cases), infants (aged 1–3 years, 18 cases), preschool (aged 4–6 years, 17 cases), school-age (aged 7–12 years, 15 cases), and adolescent groups (aged 13–14 years, 15 cases) (Table 1 ). The specimens were routinely fixed with a 4% paraformaldehyde solution (Solarbio, China), and conventional paraffin-embedded tissue sections were prepared. Paraffin-embedded archived tissues were sectioned continuously with 5 mm thickness.
4
Medical-grade Collagen-Chitosan Scaffold Fabrication
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Medical-grade collagen (Type I) with high purity was extracted from calf skin by modified method in our laboratory basing on Miller description [31 (link)]. Chitosan (CS, molecular weight (Mw): 310–375 kDa, degree of deacetylation >75%) was obtained from Sigma-Aldrich. KGM powder (purity ≥ 90%, 200-mesh) was purchased from Sheli Ltd (Hongkong, China) and further purified upon receiving. Sodium periodate (NaIO4, ≥99.5%) were provided by Aladdin (Shanghai, China). Chloral hydrate and fluorescein diacetate (FDA) were purchased from Sigma-Aldrich (St Louis, MO, USA). Phosphate buffered saline (PBS) and all sterile consumables used in cell experiments were obtained from Corning (USA). CCK-8 kits were provided by Beyotime (Shanghai, China). Bovine serum albumin 4% and paraformaldehyde solution were supplied by Solarbio (Beijing, China). The ultrapure water from a Milli-Q system (Millipore, Billerica, MA, USA) was used in all procedures of our experiments.
5
Autophagy Induction in THP-1 Cells
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THP-1 cells (1 × 105 cells/ml) transformed with the mRFP-GFP-LC3B reporter were differentiated by the use of PMA and infected with H37Ra (MOI = 10:1) in the presence or absence of sRBCs (R/M ratio = 100:1) for 24 h. A group treated with 10 nM rapamycin was used as a positive control. The cells were washed twice in PBS and fixed with 4% paraformaldehyde solution (Solarbio) for 15 min, and slides were prepared using antifade (Invitrogen), viewed under a confocal microscope (Nikon A1R), and processed using ImageJ software (NIH, USA). The level of autophagy was measured by enumerating the number of puncta per cell, and cells with >5 autolysosomes per 100 cells were counted.
6
Transwell Assay for LLC Cell Migration
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LLC cell migration was examined using transwell chambers with 8 μm pores. LLC cells (104 cells/well) suspended in serum-free medium were added to the upper transwell chambers (Thermo Fisher Scientific). Medium supplemented with 10% FBS was added to the bottom wells to stimulate migration, and the cells were incubated for 24 h. Residual cells on the upper surface of the polycarbonate membrane were removed using cotton swabs. Migrated cells on the lower surface of the chambers were fixed with 4% paraformaldehyde solution (Solarbio) for 30 min and stained with 0.5% crystal violet (Beyotime Biotechnology) for 10 min at 25°C. Images were captured using a light microscope (Leica, Wetzlar, Germany).
7
Femur and Tibia Analysis in Animal Model
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After 12 weeks, all animals were anesthetized by intraperitoneal injection of 0.3% sodium pentobarbital (Haling, Shanghai, China). Obtained the left femurs and immediately placed them in PBS (Solarbio, Beijing, China) for BMD detection. The right femurs were stored in paraformaldehyde solution (Solarbio, Beijing, China) for fixation, and the distal femoral metaphysis was selected for Hematoxylin-eosin (HE) staining. The right tibias were determined by immunohistochemistry (IHC) Staining, and the left tibias were quick-frozen with liquid nitrogen and stored at −80°C, which were used for Western Blotting (WB) and Real-time PCR (RT-PCR) detection.
8
Transwell Assay for Cell Migration
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Cell migration ability was determined by the Transwell assay. For the Transwell assay, 5 × 105 cells were seeded in the upper chambers in ECM without FBS. ECM with 20% FBS was added to the lower chambers to induce cell penetration. After 24 h of incubation, the migrated CMECs were fixed with 4% paraformaldehyde solution (Solarbio, China) and stained with crystal violet. Random visual fields of each group were imaged by an optical microscope (OLYMPUS CKX53, Japan), and the number of migrated cells was counted manually.
9
Chicken Liver Tissue Collection
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Blood samples were drawn from the left-wing vein, and then serum was isolated after incubation for 1 h at 37 °C. Chickens were stunned and killed by approved methods, then a piece of liver was snap-frozen in liquid nitrogen and stored at −80 °C for RNA extraction. Another piece of liver was fixed for 48 h in 4% paraformaldehyde solution (Beijing Solarbio Science and Technology Co., Ltd., Beijing, China) for histological analysis using hematoxylin-eosin (H&E) and oil red O staining.
10
Histological Analysis of Tissue Injury
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The paraformaldehyde solution (4%, Solarbio) was applied for tissue fixation. The organs were cut, dehydrated, and embedded to sections (4 μm). Masson trichrome (Solarbio) and H&E were applied for staining the sections. For the purpose of analyzing the injury, images were obtained from a microscope (Nikon, Japan) and determined by Image J (version 1.8.0).
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