Sections of tibiae (with tumors) were deparaffinized in xylene and degraded alcohols. Heat-induced epitope retrieval was performed by using a 2100-Retriever. Slides were rinsed with PBS, and a hydrophobic barrier was created around the tissue using a hydrophobic barrier pen (Vector Laboratories, H-4000-2). Then, slides were placed in an incubating chamber with blocking solution (Vector Laboratories, SP-6000) for 10 min and rinsed with PBS, followed by incubation with 20% horse serum (Vector Laboratories, PK-7200) for 20 min. Next, slides were incubated with the primary antibody against RFP (1:400, Abcam, ab62341, RRID: AB_945213) at 4 °C overnight and rinsed with PBS, followed by incubation with a Horse Anti-Rabbit IgG Antibody (H + L), Biotinylated, R.T.U. (Vector Laboratories, BP-1100-50) or goat IgG HRP-conjugated antibody (R&D systems, HAF017) for 30 min. After being washed again with PBS, slides were incubated with the avidin-biotin detection complex (ABC; Vector Laboratories, SK-4100) for 30 min and were then developed with 3,3′-diaminobenzidine (DAB) solution (Vector Laboratories, SK-4100). Counterstaining was performed by using Hematoxylin QS (Vector Laboratories, H-3404). Slides were scanned with an Aperio CS2 Digital Pathology Slide Scanner (Leica Biosystems).
Sk 4100
Manufactured by Vector Laboratories
Sourced in United States, United Kingdom, Canada
The SK-4100 is a high-performance spectrophotometer designed for precise and reliable absorbance measurements. It features a wide wavelength range, adjustable bandwidth, and a digital display for accurate readouts.
Automatically generated - may contain errors
Lab products found in correlation
Pk 6100, by Vector Laboratories (27 mentions)
Ba 1000, by Vector Laboratories (23 mentions)
Pk 4000, by Vector Laboratories (12 mentions)
Vectastain elite abc kit, by Vector Laboratories (10 mentions)
H 3300, by Vector Laboratories (9 mentions)
Pk 6101, by Vector Laboratories (8 mentions)
Vectastain abc kit, by Vector Laboratories (6 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (6 mentions)
Ba 9200, by Vector Laboratories (5 mentions)
Pk 6200, by Vector Laboratories (5 mentions)
Sa 5004, by Vector Laboratories (4 mentions)
H 3401, by Vector Laboratories (4 mentions)
S 1000, by Vector Laboratories (4 mentions)
Axiovision release 4, by Zeiss (3 mentions)
Ab152, by Merck Group (3 mentions)
Ba 2000, by Vector Laboratories (3 mentions)
Tcs sp8, by Leica (3 mentions)
Testicular hyaluronidase, by Merck Group (3 mentions)
Sp 5030, by Vector Laboratories (3 mentions)
Mhs16, by Merck Group (3 mentions)
221 protocols using sk 4100
1
Immunohistochemical Analysis of RFP Expression
2
Immunohistochemical Analysis of Lung Tissues
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Immunohistochemistry was carried on the paraffin sections of the left lung as per standard protocol of our lab [25 (link)]. The sections were processed and incubated with primary antibodies against TLR-4 (sc12511; Santa Cruz; dilution 1:400), IL-1β (sc-1252, Santa Cruz; dilution 1:200) and TNF-α (sc1350; dilution 1:2000) for 1 hour followed by a suitable secondary antibody (Dako P0449; dilution 1:800) for 30 min. Color development was done with a commercial kit (SK4100; Vector Laboratories, USA) followed by counter staining with haematoxylin.
3
Immunohistochemical Staining of Cartilage
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IHC staining of GFP-positive cells was used to reveal donor cells in the regenerated cartilage and Col2 staining was used to detect specific cartilage matrix collagen 2. Briefly, after deparaffinization, washing, and blocking with 5% donkey serum in PBS, sections were incubated with rabbit anti-GFP antibody (ab290, Abcam, 1:1000 dilution) and rabbit anti-Col2 (ab34712, Abcam: 1:400 dilution) in 5% donkey serum overnight. For Col2 staining, antigen retrieval was performed using 2% hyaluronidase (H3506-5G, Sigma,) in PBS at room temperature for 30 min, followed by washing with PBS three times before blocking and incubation with primary antibody. The following day, sections were treated with 0.5% H2O2 in PBS for 30 min at room temperature, washed in PBS, and then incubated with goat anti-rabbit biotin (BA 1000, Vector Laboratories, Burlingame, CA, USA, 1:200 dilution) for 2 h at room temperature. After three washes, each slide was incubated with ABC reagent (PK 7200, Elite ABC kits, Vector Laboratories) for 2 h at room temperature. After three washes with PBS, diaminobenzidine (DAB) staining (SK-4100, Vector Laboratories) was used to visualize the GFP-positive cells. Hematoxylin (H-3404, Vector laboratories) counterstaining was performed following the DAB color reaction.
4
Histological Analysis of Murine Bone
5
Immunohistochemical Analysis of Substantia Nigra
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Newborn pups were dislocated and fix in 4% PFA for 6 h. Two- and 10-month-old mice were anesthetized by intraperitoneal (i.p.) injection of chloral hydrate (400 mg/kg) (Sigma-Aldrich) and were perfused transcardially using a 4% PFA Immunofix solution (05-K01015, Bio-optica, Milan, Italy). After 4 h post-fixation, brains were incubated in a solution of PBS with high salt concentration (NaOH 200 mM, NaH2PO4 245 mM, NaCl 0.9%) containing 18% sucrose for at least 24 h, then 30 µm coronal sections were cut with a cryostat (Leica Biosystems, Milan, Italy) and conserved in 60% glycerol. Sections of the Substantia Nigra (SN) and striatum were permeabilized with 20% methanol and 5% H2O2, washed and incubated with primary antibody (TH, AB152, Merck Millipore) overnight (ON) at 4 °C. On the following day, sections were washed and probed with anti-rabbit biotinylated secondary antibody for 45 min at rt. This was followed by gentle washing, incubation with avidin–biotin complex (PK6100, ABC kit, Vector Laboratories, Burlingame, CA, USA) at rt for 45 min and 3,3′-diaminobenzidine (DAB) staining (SK-4100, Vector Laboratories) for 5 min. Finally, sections were washed, dehydrated, mounted with Vectamount mounting medium (H-5000-60, Vector Laboratories) and were observed by means of an inverted light/epifluorescence microscope (Olympus IX50; Olympus, Milan, Italy).
6
Immunohistochemical Analysis of SV2A and SV2B
7
Immunohistochemical Analysis of SV2A and SV2B
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Immunohistochemical staining for SV2A and SV2B were completed at 14 days post-injury. Immunohistochemical staining was completed in 24 well plates on free-floating tissue sections utilizing one section from each animal, for each isoform, focused at the CCI injury site incorporating bregma levels −3.2 to −4.0 mm [34 ]. The sections were rinsed with 0.1M tris-buffered saline (TBS) buffer and blocked with 10% normal goat serum and 0.1% Triton X-100 in 0.1M TBS (TBS-T) for 1 hour. Sections were incubated overnight at 4°C with anti-SV2A antibody (rabbit polyclonal 1:1,000, SySy, Cat. No. 119003) or anti-SV2B antibody (rabbit polyclonal 1:1,000, SySy, Cat. No. 119103). The following day, sections were rinsed with TBS-T, incubated with horseradish peroxidase secondary antibody and rinsed with TBS-T to prepare for substrate development. For each isoform, all sham control and CCI-injured tissues were run simultaneously, diaminobenzidine (DAB) was added, and the reaction development time was tightly controlled to ensure equal diaminobenzidine (DAB) substrate exposure time between all sections within each isoform set for measurements of pixel intensity (SK-4100, Vector Laboratories, Burlingame, CA). Sections were mounted on Superfrost Plus slides (Fischer Scientific), and were cover-slipped using Permount medium (Fischer Scientific).
8
Immunolocalization of Canine Germ Cell Markers
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To identify the localization of PGP9.5-, SCP3- and acrosin-expressing cells in the dog testes, the antigens were retrieved by boiling the tissue for 30 minutes in Tris-ethylenediaminetetraacetic acid (Tris-EDTA) solution (10 mM Tris base, 1 mM EDTA, and 0.05% Tween-20; pH 9). The membranes of the antigen-retrieved tissues were permeabilized with phosphate-buffered saline (PBS) containing 0.2% Triton X-100 for 10 minutes. Non-specific protein binding was blocked with 2% bovine serum albumin in PBS for 1 hour at room temperature (RT). Tissues were incubated overnight at 4℃ with the following primary antibodies: PGP9.5 (1:1000 dilution; 7863-1004; AbD Serotec; Raleigh, NC, USA), SCP3 (1:50 dilution; SC-33195; Santa Cruz Biotechnology, Inc.) and acrosin (1:50 dilution; SC-51504; Santa Cruz Biotechnology, Inc.). After the samples were washed three times with PBS, the appropriate secondary antibody was added and the samples were incubated for 2 hours at RT. A peroxidase substrate detection kit (SK-4100; Vector Laboratories; Burlingame, CA, USA) was used for the detection of putative canine germ cell markers, according to the manufacturer's instructions. Finally, DPX mountant (Sigma Aldrich, 06522) was used to fix the immunostained canine testicular tissues. The immunostained tissues were observed under an optical microscope.
9
SARS-CoV-2 Nucleocapsid Protein Immunohistochemistry
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Immunohistochemistry was performed as we previously described with slight modifications31 . Human lung tissue samples were collected and fixed overnight in 10% formalin. The fixed samples were embedded in paraffin by TP1020 Leica semi-enclosed benchtop tissue processor and sectioned with microtome (Thermo Fisher Scientific). Sectioned samples were prepared on glass slides and were dewaxed and dehydrated by serially diluted xylene, ethanol, and double-distilled water in sequence. Afterwards, the samples were co-heated together with antigen unmasking solution (H-3300, Vector Laboratories) at 85 °C for 90 s for antigen exposure, followed by blocking with 0.3% hydrogen peroxide for 30 min, and 1% BSA for 30 min. The in-house rabbit anti-SARS-CoV-2-N immune serum or in-house rabbit anti-SARS-CoV-N immune serum were applied as the primary antibodies and were incubated with the slides at 4 °C overnight. The signal was developed with the DAB (3,3’-diaminobenzidine) substrate kit (SK-4100, Vector Laboratories). Cell nuclei were labeled with Gill’s haematoxylin. The slides were mounted with antifade mounting medium with DAPI (H-1200, Vector Laboratories). Images were taken with the Olympus BX53 light microscope (Olympus Life Science).
10
Immunostaining for Protein Detection
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For immunostaining, the slides were incubated in 0.3% H2O2 diluted in 100% MeOH for 30 min to block endogenous peroxidase activity. The sections were incubated at room temperature for 30 min in PBS containing 1% BSA (Sigma, 9048-46-8) to block nonspecific binding and then incubated overnight at 4 °C in a humidified chamber with an antibody against His-tag (Affinity, T0009) or α-SMA diluted 1:500 in PBS containing 1% BSA. Then the slides were incubated for 30 min at room temperature in a humidified chamber with anti-mouse IgG antibody (Vector Laboratories, BA–9200) and then HRP conjugated streptavidin (Boster, BA1088) in PBS. Then the slides were incubated with 50 μl of diaminobenzadine (Vector Laboratories, SK4100) as a substrate, counterstained with hematoxylin (LEAGene, DH0006), dehydrated, and fixed with permount histological neutral balsam.
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