To assess in situ vascular cell proliferation in patients with IPAH, proliferating cell nuclear antigen (PCNA) staining was performed. Tissue sections were deparaffinized in toluene, treated with a graded series of ethanol washes, and rehydrated in PBS (Sigma Aldrich, St. Louis, MO, USA). Slides were then incubated for 30 min in a protein blocking solution (10% goat serum in PBS) and incubated for 1 h with an anti-PCNA mouse monoclonal antibody (M0879, clone PC-10, 1:200; Dako, Santa Clara, CA, USA) in the presence of streptavidin/biotin endogenous blocking reagents (SP-2002; Vector, Burlingame, USA). The slides were then incubated with a mouse biotinylated secondary antibody for 30 min, followed by amplification with the Vectastain ABC-AP Kit (AK-5002; Vector) for 30 min. The slides were then processed using the Vector Red Alkaline Phosphatase Substrate Kit (SK-5100; Vector).
Sp 2002
Manufactured by Vector Laboratories
Sourced in United States
The SP-2002 is a high-performance spectrophotometer designed for a wide range of applications. It features a compact and robust design, providing accurate and reliable measurements across a broad wavelength range.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780 confocal microscope, by Zeiss (3 mentions)
D1306, by Thermo Fisher Scientific (2 mentions)
T8787, by Merck Group (2 mentions)
B 1325, by Vector Laboratories (2 mentions)
Fluoromount g, by Southern Biotech (2 mentions)
B 1085, by Vector Laboratories (2 mentions)
Streptavidin a488, by Thermo Fisher Scientific (2 mentions)
Dmi8 inverted microscope, by Leica (2 mentions)
Vectashield mounting medium h 1000, by Vector Laboratories (2 mentions)
Tissue tek cryomold and oct gel compound, by Sakura Finetek (2 mentions)
Peanut agglutinin biotin b 1075, by Vector Laboratories (2 mentions)
Af647 sav, by Thermo Fisher Scientific (2 mentions)
Cd3 fitc 17a2, by Thermo Fisher Scientific (2 mentions)
Cd45r b220 ra3 6b2, by BD (2 mentions)
Af555 goat anti rat igg, by Thermo Fisher Scientific (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Vectastain abc ap kit, by Vector Laboratories (1 mentions)
Vector red alkaline phosphatase substrate kit, by Vector Laboratories (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Alexa fluor 488 conjugated streptavidin, by Vector Laboratories (1 mentions)
10 protocols using sp 2002
1
Quantifying PCNA-Positive Cells in IPAH
2
Immunostaining Procedure for Fluorescent Microscopy
3
Immunofluorescence Staining of Murine Spleen
4
Germinal Center Immunostaining in Frozen Mouse Spleen
5
Immunofluorescence Staining of Kidney Organoids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Kidneys organoid and LTL+ cells were washed with PBS. Next samples were fixed with 4% paraformaldehyde (153799, Aname) for 20 min at room temperature. Specimens were washed twice with PBS and further blocked using Tris-buffered saline (TBS) with 6% donkey serum (S30, Millipore) and 1% Triton X-100 (T8787, Sigma) for 1h at room temperature. After three rinses with antibody dilution buffer, samples were treated for 4h at room temperature with fluorescent conjugated secondary antibodies (Alexa Fluor (A) Cy3- or A647-; 1:200). A previous blocking step with a streptavidin/biotin blocking kit (SP-2002, Vector Labs) was performed for biotinylated LTL (B-1325, Vector Labs) and Alexa Fluor 488-conjugated streptavidin (SA5488, VectorLabs) to detect LTL+ cells. Antibodies to NEPHRIN (R&D SYSTEMS 4269; 1:100) and LAMININ (Sigma L9393; 1:50), SGLT2 (Abcam AB37296; 1:100), NaKATPase (Abcam; AB209299; 1:200) and SLC3A1 (Sigma HPA038360; 1:50) were used overnight at 4°C diluted in antibody dilution buffer consisting of TBS with 6% donkey serum and 0.5% Triton X-100. Nuclei were detected using 4,6-diamidino-2-phenylindole (DAPI; 1:5000, D1306, Life Technologies) for 30min. For mounting, samples were immersed in Fluoromount-G (0100-01, Southern Biotech). Sample confocal images were acquired with an SP5 Leica microscope and LTL + were analyzed using ImageJ.
6
Immunohistochemical Visualization of Glycoconjugates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Coronal sections were washed three times for 10 minutes in PBST. For wisteria floribunda agglutinin (WFA) staining, samples were first blocked using a biotin blocking kit (Vector Labs SP2002). Sections were then incubated in block (PBST +5% Normal Goat Serum (NGS)(Thermo PCN5000), +5% Normal Donkey Serum (NDS)(Sigma D9663)) for 1 hour and then incubated in primary antibody solution(PBST + 2.5% NGS and 2.5% NDS) overnight at 4 °C. Primary antibodies used were chicken anti-GFP (1:500 Abcam ab13970), mouse anti-CS-56 (1:200 Sigma C8035), biotinylated WFA (1:1000 Vector Labs B-1355). Sections were washed three times for 10 minutes in PBS and then incubated for 1 hour at RT in secondary antibody solution (0.5x blockPBST +2.5% NGS and 2.5% NDS). Secondary antibodies used were goat anti-chicken Alexa 488 (1:1000 Invitrogen A11039), 10 μM HTL646 (1:500), Streptavidin-cy3 (1:1000 Jackson Immuno Research 016-160-084) and Hoescht 33343 (1:10K Invitrogen H3570). sections were washed three times for 10 minutes in PBS, and then placed on slides. Sections were mounted with flouromount gold and coverslips (TedPella 260152) and left to dry overnight at RT and stored at −20 °C.
7
Immunofluorescent Staining of Murine Spleen
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cryosections (4μm) of spleens from Nr2f6+/+ and Nr2f6−/− immunized mice were fixed with 1% paraformaldehyde for 10 min at room temperature (RT). Slides were then blocked with 2% BSA/PBS for 30 min at RT, followed by incubation with mouse monoclonal antibodies against CD3 (eBioscience 17-0031-82, APC), B220 (eBioscience 12-0425-82, PE) and a biotin-labeled PNA (Vector Labs B-1085) for 45 min at RT. A kit for endogenous biotin and avidin blocking was used according to the manufacturer’s instructions (Vector Labs SP-2002). Subsequently, slides were incubated with streptavidin A488 (Invitrogen S32354) for 45 min at RT. After washing, slides were mounted with Diamond Antifade medium (Invitrogen P36965), images were acquired on a Leica DMi8 inverted microscope, and processed using Fiji software (Schindelin et al., 2012 (link)).
8
Immunostaining of Human Islet and Mouse Pancreas
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human pseudo islets were fixed for 1 h at 4 °C and embedded in collagen (Wako Chemicals), mouse pancreata were fixed in 4% paraformaldehyde overnight at 4 °C. Ten µm-thick frozen sections were cut and stained following standard cryostaining protocols. Briefly, sections were washed in PBS, incubated with blocking solution (Vector Labs, SP-2002) at room temperature, followed by incubation in permeabilization/blocking buffer (1% bovine serum albumin, 0.2% non-fat milk, 0.5% Triton-X in PBS) for 1 h. Primary antibodies were mixed with permeabilization/blocking buffer at appropriate concentrations and slides were incubated at 4 °C overnight. The following primary antibodies were used: Guinea pig anti-Insulin, (1:1000, DAKO, A0564), rabbit anti-BCL11A (1:1000, Bethyl Laboratories, A300–380), mouse anti-mCherry (1:500, Abcam, ab125096), goat anti-Glut2 (1:300, Santa Cruz Biotechnology, sc-7580). Slides were washed with PBS, incubated with secondary antibodies at room temperature for 2 h. Following the final wash with PBS, slides were preserved with mounting medium containing DAPI (Vector Labs, Vectashield H-1200). Images were obtained using a Leica SP2 confocal microscope.
9
Multiplex Immunohistochemistry Staining Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissues were treated with the epitope retrieval protocol. Samples to be treated with a cocktail containing a biotinylated secondary were incubated with a streptavidin and biotin blocking solution (Vector Laboratories; Catalog No SP-2002). All tissues were blocked with Serum-Free Protein Block for 30 min. Samples were subsequently incubated in a cocktail mixture of primary antibody solution for 60 min then rinsed with ImmunoWash. A cocktail mixture of secondary antibodies against the primary antibody host species was applied to the tissues for 30 min. The secondary antibodies (Supplementary Table 2 ) that were used in the study include the following: donkey anti-rabbit (Thermo Fisher Scientific; Catalog No: A21206, Dilution: 1:1000), horse anti-rabbit (Vector Laboratories; Catalog No: DI-1088, Dilution: 1:300), biotinylated donkey anti-mouse (Thermo Fisher Scientific; Catalog No: A16021, Dilution: 1:500), biotinylated horse anti-mouse (Vector Laboratories; Catalog No: BA-2001, Dilution: 1:75), biotinylated donkey anti-rat (Novus Biologicals; Catalog No: NBP1-75379, Dilution 1:500), and biotinylated donkey anti-goat (Thermo Fisher Scientific; Catalog No: A16009, Dilution: 1:500). This was followed by an incubation with a streptavidin conjugate for 30 min. Slides were counterstained with DAPI.
10
Germinal Center Detection in Immunized Mouse Spleens
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Spleens from immunized mice were froZen in optimal cutting temperature (OCT) compound (Tissue-Tek cryomold and OCT gel compound, Sakura Finetek USA, Torrance CA) and cryosectioned in 5μm slices. Cryosections were fixed in cold acetone for 10 min at -20°C, washed with PBS, and then solubilized in 0.5% Tween-20 in PBS. After further washing in PBS, endogenous biotin and streptavidin binding sites were blocked according to manufacturer's instructions (SP-2002, Vector Laboratories, Burlingame CA). For germinal center detection, sections were sequentially incubated with the following primary antibodies for 30 min at RT: CD3-FITC (17A2, eBioscience, San Diego CA, 1:100); CD45R/B220 (RA3-6B2, BD, Franklin Lakes NJ, 1:200); peanut agglutinin-biotin (B-1075, Vector Laboratories, Burlingame CA, 1:100). Slides were washed with 0.1% Tween-20 in PBS, and then incubated with secondary antibodies for 30 min at room temperature (AF555-goat anti-rat IgG (Invitrogen, Carlsbad CA, 1:500); AF647-sAv (Invitrogen, Carlsbad CA, 1:200)). After washing, slides were mounted with Vectashield Mounting Medium (H-1000, Vector Laboratories, Burlingame CA). Images were acquired on a Zeiss LSM 700 Confocal Microscope (magnification 200x) and pictures processed using Zen (Zeiss, Oberkochen Germany) and QuPath software.
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!