Timm’s staining was performed as previously described (Wang et al., 2016 ). Mice were anesthetized and perfused intracardially with 50 ml 0.9% NaCl (Sigma-Aldrich) solution, followed by 50 ml 4% paraformaldehyde solution (Sigma-Aldrich), 100 ml 1% sodium sulfide solution (Sigma-Aldrich), and 50 ml 4% paraformaldehyde solution again. Slices were stained in Timm’s developer solution for 110 min in darkness at room temperature. Timm’s developer solution was freshly made from 60 ml gum arabic (30 g; Vetec), 10 ml citric acid monohydrate (2.55 g; Beijing Chemical Works) and trisodium citrate dehydrate (2.35 g; Beijing Chemical Works), 15 ml hydroquinone (0.85 g; Sigma-Aldrich), and 15 ml silver nitrate (0.11 g; Sinopharm Chemical Reagent). Subsequently, sections were washed with distilled water and counterstained with hematoxylin (ZSGB-BIO). An investigator blinded to the treatment assessed the degree of mossy fiber sprouting into the supragranular layer of the DG. The standard for scoring was described previously (Wang et al., 2016 ).
Paraformaldehyde solution
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Italy
Paraformaldehyde solution is a fixative commonly used in various laboratory applications. It is a stabilized, aqueous solution of paraformaldehyde, a polymer of formaldehyde. Paraformaldehyde solution is used for preserving and fixing biological samples, such as cells and tissues, prior to further analysis or processing.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (28 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (9 mentions)
Hoechst 33342, by Thermo Fisher Scientific (8 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (5 mentions)
Tritc conjugated phalloidin, by Merck Group (5 mentions)
Hoechst 33342, by Merck Group (4 mentions)
Fitc phalloidin, by Merck Group (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Donkey serum, by Merck Group (3 mentions)
Sucrose, by Merck Group (3 mentions)
Phosphate buffered saline (pbs), by Takara Bio (3 mentions)
Trypsin, by Thermo Fisher Scientific (3 mentions)
Crystal violet, by Solarbio (3 mentions)
Dapi 4 6 diamidino 2 phenylindole, by Merck Group (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Lsm 780, by Zeiss (3 mentions)
4 6 diamidino 2 phenylinodole dapi, by Merck Group (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
155 protocols using paraformaldehyde solution
1
Timm's Staining for Mossy Fiber Sprouting
2
Immunofluorescence Staining of Flavivirus Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were fixed in 4% paraformaldehyde solution (PFA; Sigma-Aldrich, St. Louis, MO, USA) in phosphate-buffered saline (Thermo Fisher Scientific), permeabilized with PBS/0.1% Triton X-100 (Sigma-Aldrich) and blocked in 4% bovine serum albumin (BSA, Sigma-Aldrich) in PBS. Then, cells were incubated with the primary antibodies diluted in PBS with 4% BSA. Primary antibodies specific for Flavivirus envelope protein E (clone 4G2, mouse, 1:500, Merck Millipore, USA), PAX6 (rabbit, 1:100, Sigma-Aldrich), Nestin (mouse, 1:100, Abcam, Cambridge, UK) were used. Cells were incubated overnight at 4 °C. The secondary antibodies used included the anti-mouse IgG Alexa Fluor-488 (goat, 1:250, Thermo Fisher Scientific) and the anti-rabbit IgG Alexa Fluor-546 (goat, 1:250, Thermo Fisher Scientific). DRAQ5 fluorescent probe solution (Thermo Fisher Scientific) was used to stain nuclei. Immunofluorescence was visualized by a confocal microscope (Leica, Wetzlar, Germany) under 63× magnification.
3
Quantifying DNA Damage in B Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Following the overnight exposure of the PBMCs to the CM (as described above), the PBMCs were collected and diluted at 5 × 105 cells/mL. Then, 50 µL of the cell suspension (25 × 103 cells) was transferred into flow cytometry tubes, DPBS + 2% FBS was added, and the cells were centrifuged for 5 min at 500× g at room temperature (RT). The cells were then fixed with a 1.5% paraformaldehyde solution (Sigma-Aldrich) for 20 min at RT, followed by a second wash with DPBS + 2% FBS. After the fixation step, the cells were permeabilized by exposing them to 90% methanol for 20 min at 4 °C. After another wash with DPBS + 2% FBS, the cells were stained with 5 µL of an Alexa Fluor® 488-conjugated, γH2AX antibody (Clone N1-431; BD Biosciences) and 20 µL of an APC-conjugated, CD19 antibody (BD Biosciences) for 20 min at RT. A final wash with DPBS + 2% FBS was performed before acquiring the data with the BD Accuri™ C6 flow cytometer (BD Biosciences). The gating strategy was as follows: first, all CD19+ cells were selected in a dot plot, and γH2AX+ cells were then identified in this CD19+ population.
4
Fluorescent Imaging of MG63 Cytoskeleton
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MG63 cells were seeded at 15 × 103 cell/cm2 on a glass coverslip in a 12 well-plate and incubated for 24 h. Then, cells were treated with the different compounds at their IC50 values. After 24 h, cells were washed twice with PBS and fixed for 15 min in a 4% (w/v) paraformaldehyde solution (Sigma-Aldrich Chemie GmbH, Schnelldorf, Germany) at room temperature. After three washes with PBS, cells were stained for 45 min at room temperature with FITC-phalloidin (Sigma-Aldrich Chemie GmbH, Schnelldorf, Germany) and Hoechst 33342 (Sigma-Aldrich Chemie GmbH, Schnelldorf, Germany). FITC-phalloidin and Hoechst 33342 were solubilized in PBS at a final concentration of 1µg/mL. Finally, sample were washed and mounted with Mowiol® 4-88 (product reference, 81381; Sigma-Aldrich Chemie GmbH, Schnelldorf, Germany) containing DABCO as antifading. Images were acquired with a Nikon eclipse 90i fluorescence microscopy (Nikon Corporation, Tokyo, Japan).
5
Immunocytochemical Characterization of Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Each small aliquot of cells was cultured in a 24-well plate for 5 days. Then, the cells were washed in phosphate-buffered saline (PBS, Sigma-Aldrich, Milwaukee, WI, USA) and fixed in a 4% paraformaldehyde solution (Sigma-Aldrich, Milwaukee, WI, USA) for 10 min; 3% peroxidase eliminated endogenous hydrogen peroxide; and CD105, CD34, CD45 (1 : 10) (BD Biosciences, San Jose, CA) with fluorescent antibody were added after extensive washing with PBS. Cells were incubated in 5% CO2 at 37°C for two hours. Cells were mounted in glycerol/buffer on a glass slide after extensive washing with PBS. Images of the labeled cells were obtained using a fluorescence image restoration microscope (Applied Precision, USA).
6
Quantifying Cartilage Proteoglycan Content
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All cartilage specimens were processed as follows: samples were fixed in 4 % paraformaldehyde solution (Sigma) for 36 h and then decalcified by immersion in 40 % EDTA-2Na solution (Solarbio) for 2 weeks. The full-thickness tissues were then rip-cut sectioned into 8-mm slices and stained with Safranin-O stain (Sigma) to determine PG content. Stained samples were observed under a microscope (IX-71, OLYMPUS, Tokyo, Japan) and photographed. For each specimen, 10 random non-overlapping fields of view were observed. Image-Pro Plus 6.0 software (Media cybernetics, MD, USA) was used to determine the integrated optical density (IOD) of the Safranin-O stain intensity, to quantitatively determine PG content.
7
Immunofluorescence Staining of Cultured Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunofluorescence, cells grown on coverslips were fixed by incubation with 4% (w/v) paraformaldehyde solution (Sigma-Aldrich) for 10 min at room temperature and permeabilised by incubation with PBS containing 0.2% (v/v) Triton X-100. This was followed by incubation with the desired primary antibody made up in 3% (w/v) bovine serum albumin, 0.2% (v/v) Triton X-100 in PBS for 1 hr. Following washing in 0.2% (v/v) TX-100 cells were incubated with secondary antibody anti-mouse Alexa Fluor 488 (RRID:AB_2534069 ) or anti-rabbit Alexa Fluor 568 (RRID:AB_2534078 ; Molecular Probes). For nuclear staining, cells were incubated with 0.01 mg/ml Hoechst 33258 (Sigma-Aldrich) in PBS for 5 min following incubation with secondary antibodies. Cells were analysed using Deltavision DV Elite Imaging System (Applied Precision) and then deconvolved images were analysed using ImageJ.
8
F-Actin Staining for Cell Adhesion Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To evaluate cell adhesion, F-actin filaments were stained 48 h after seeding. Cells were fixed for 1 h with a 4% paraformaldehyde solution (Sigma Aldrich Co, Steinheim, Germany). After fixation, the cell membrane was permeabilized for 1 h with 0.01% Triton X-100 solution (Sigma Aldrich Co, Steinheim, Germany) in bovine serum albumin (BSA). To diminish scaffolds’ affinity to fluorescence, cell–scaffold systems were incubated for 40 min with a 0.04% Trypan Blue solution, which was then thoroughly washed with PBS. Composites were incubated overnight with phalloidin–FITC (Sigma Aldrich Co, Steinheim, Germany) and for 20 min with Hoechst 33342 for cell nuclei staining (ThermoFisher Scientific, Foster City, CA, USA). Laser-scanning confocal microscope was also used for visualization and the images inspection was performed using microscope’s software. Additional details of cell morphology and spreading were acquired using the ESEM equipment described in Section 3.3.5 .
9
Immunofluorescence Imaging of Flavivirus
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells grown on the cover-slip were fixed for 20 min in 4% paraformaldehyde solution (Sigma) in phosphate-buffered saline (PBS, Euroclone). Cells were permeabilized for 30 min in blocking solution, containing 0.2% Triton X-100 (Sigma) and 10% donkey serum (Sigma) and incubated overnight at 4 °C with the primary mAb in blocking solution. The following mAbs specific for Flavivirus E protein (1:200, Millipore, MAB10216), double-stranded RNA (1:300, English and Scientific Consulting Kft, Hungary), vimentin (1:300, Bioss, BS-0756R) and calreticulin (1:300, Sigma, C4606) were used. Cells were then washed with PBS and incubated for 1 h with Hoechst and either anti-mouse Alexa Fluor-488 or anti-rabbit Alexa Fluor-594 secondary Abs (1:1,000 in blocking solution, ThermoFisher Scientific). High resolution wide field fluorescence images were acquired on a Nikon Eclipse Ni-U microscope equipped with a Nikon 60x plan apo 1.40 oil or a 20x plan apo 0.75 objective and a Nikon DS-Qi2 camera.
10
Quantifying Angiogenic Potential of HUVECs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The 96-well culture plates were coated and incubated with 10 mg/ml growth factor-reduced Matrigel solution (BD Biosciences, San Jose, CA, USA) and polymerized at 37°C for 30 mins. HUVECs were harvested and added to Matrigel-coated plates with or without compounds. After 24 h, HUVECs were fixed with 4% paraformaldehyde solution (Sigma Aldrich, St. Louis, MO, USA) and stained with Mayer's hematoxylin (Muto Pure Chemicals, Tokyo, Japan) [16 (link)]. The tubular structure formation in HUVECs was photographed under a phase-contrast microscope at 200x magnification. Tube length was quantified by using the ImageJ software (National Institutes of Health, Bethesda, MD, USA). Data were expressed as the percentage of incremental increase of the total tube length in HUVECs.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!