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).
Pk 7200
Manufactured by Vector Laboratories
Sourced in United States
The PK-7200 is a versatile lab equipment product from Vector Laboratories. It is designed to perform a core function within the laboratory setting. The details of its intended use are not provided in this factual and unbiased description.
Automatically generated - may contain errors
Lab products found in correlation
Sk 4100, by Vector Laboratories (2 mentions)
Ab62341, by Abcam (1 mentions)
Hematoxylin qs, by Vector Laboratories (1 mentions)
Haf017, by R&D Systems (1 mentions)
Aperio cs2 digital pathology slide scanner, by Leica (1 mentions)
Sp 6000, by Vector Laboratories (1 mentions)
Hydrogen peroxide, by Merck Group (1 mentions)
Sp 2001, by Vector Laboratories (1 mentions)
Novared, by Vector Laboratories (1 mentions)
Anti active caspase 3, by R&D Systems (1 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (1 mentions)
Normal horse serum, by Vector Laboratories (1 mentions)
S 2012 50, by Vector Laboratories (1 mentions)
Dv2004g1, by Biocare Medical (1 mentions)
H 3404, by Vector Laboratories (1 mentions)
Ab34712, by Abcam (1 mentions)
Abc elite kit, by Vector Laboratories (1 mentions)
Ab290, by Abcam (1 mentions)
Ba 1000, by Vector Laboratories (1 mentions)
Eclipse ti, by Nikon (1 mentions)
8 protocols using pk 7200
1
Immunohistochemical Analysis of RFP Expression
2
Immunohistochemical Analysis of Apoptosis and ERK Activation in Tumor Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Pieces of the tumours were fixed for 12 h in 3.7% formaldehyde in PBS, followed by 48 h in 70% Ethanol. Standard histological processing and paraffin embedding were done. Sections of 3 μm thickness from paraffin-embedded tissues (PET) were heated to 56°C, rehydrated, washed and treated with TE (Tris 10 mM, EDTA 1 mM pH9) 30 min at 98°C for antigen retrieval. Endogenous peroxidases were inhibited with 3% hydrogen peroxide (Sigma) in H2O for 5 min. Non specific sites and avidin were blocked with buffers from kit PK-7200 and SP-2001 from Vector laboratories (CA, USA). Slides were incubated with primary anti-Active caspase3 1:300 (AF835 R&D Systems Minneapolis, MN, USA) or anti-phospho-ERK1/2 (Cell Signaling) for 1 hour followed by 30 min incubation at room temperature with biotinylated secondary antibodies (SP-2001 Vector Laboratories) and revealed by an additional 10 min incubation with Novared (SK-4800 Vector Laboratories). Slides were counterstained with hematoxylin, dehydrated and mounted with Eukitt-mounting medium (Labonord).
3
Lung Adenoma Proliferation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
5 μm lung sections from urethane, iloprost, and saline groups were deparaffinized, blocked with 0.3% hydrogen peroxide, and antigen retrieval performed in boiling Diva Decloaker (Biocare Medical, DV2004G1) under pressure for 5 minutes. Sections were blocked with Background Punisher (Biocare Medical BP974M) and 2.5% Normal Horse serum (Vector Labs S-2012-50). Sections were incubated in Ki67 primary Ab (1:2000 dilution, Abcam 15580) for 1 hour at room temperature, followed by anti-rabbit universal antibody for 30 minutes and ABC reagent for 30 minutes at room temperature (Vector Laboratories PK-7200). Ki67+ nuclei were detected using Betazoid DAB chromagen kit (Vector Laboratories BDB2004). Tumor area was measured and Ki67-positive nuclei/mm2 were counted in each tumor. Replicate blinded counts were conducted. The Ki67+ nuclei/mm2 tumor area for each tumor was averaged by group (Dwyer-Nield et al., 2017 (link)). H&E stains were done on 5 μm lung sections from urethane, iloprost, and saline groups and were used to confirm adenoma presence and structure.
4
Immunohistochemical Staining of Cartilage
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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.
5
Immunohistochemical Analysis of Apoptosis and Proliferation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Paraffin sections were deparaffinized in xylene and rehydrated through a gradual decrease in ethanol concentration. Antigen epitopes were then unmasked using sodium citrate buffer (pH 6.0). Subsequently, the sections were incubated overnight at 4°C using the following primary antibodies: anti-GFP (#2555; Cell Signaling Technology), anti-Ki-67 (ab15580; Abcam, Cambridge, UK). After primary antibody incubation, sections were incubated with a biotinylated anti-rabbit IgG secondary antibody (PK-7200; Vector Laboratories, Burlingame, CA, USA) followed by treatment with freshly prepared DAB substrates (PK-4100; Vector Laboratories). Sections were lightly counter-stained with hematoxylin and mounted. Apoptosis was assessed in liver sections using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) staining (ApopTag® In Situ Apoptosis Detection Kits; Merck, Billerica, MA, USA). Slides were analyzed and photographed using a microscope (Eclipse Ti; Nikon, Tokyo, Japan) equipped with a digital camera.
6
Immunohistochemical Analysis of Ki-67
7
Immunohistochemical Detection of p16
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cases were evaluated for the detection of p16 by immunohistochemistry employing an ABC system (Vector Laboratories, Burlingame, USA). Antigen unmasking was carried out at 120°C for 3 min in a pressure cooker with a TRIS-EDTA buffer (1,2 g/L Tris, 0,36 g/L EDTA, pH 9.0). Sections were pre-treated for 10 min with 1% H 2 O 2 in 0.1 M phosphate buffered saline (PBS), pH 7.4 to quench endogenous peroxidase activity, and blocked for 30 min at room temperature (rt) in PBS with a 2% normal horse serum (PK-7200, Vector Laboratories, Burlingame, USA) and 0.05% TritonX-100. Sections were then incubated overnight at 4˚C with a 1:100 diluted monoclonal primary antibody Sampling material for q-PCR Three 10 µm thick sections from the selected samples were cut with a microtome, place onto slides and left unfixed. These slides were observed under an optic microscope. Tissue that was not relevant for the study was scraped off and the remaining tissue directly collected in a DNase free 1,5 ml tube. To prevent carryover of contaminating DNA the microtome overlay was covered with a new piece of adhesive tape and a new blade was used for each sample.
8
Immunohistochemical Staining Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The study material, embedded in paraffin, was cut serially at 5 µm slices on the rotary microtone (Leica) into silane-coated glass slides (Silan Sigma A36648), deparaffinized in xylene 3 × 10 min, hydrated in an ethyl alcohol series, 100%, 96% and 70% for 10 min and rinsed in distilled water for 10 min. Epitopes were exposed to temperature of 100 o C 3 × 5 min in citrate buffer (pH 6.0), cooled at room temperature for 10 min and rinsed in distilled water for 10 min. These were followed by blocking of endogenous peroxidase with 0.3% TRIS + H 2 O 2 (TrisBase T1503, pH 7.4-7.6) for 10 min and rinsed in TRIS for 10 min and blocked with 2.5% normal horse serum (Vector PK-7200) for 1 h. Specimens were incubated with GFAP antibody (1 : 700) (MCA 9733 G-1, Bio-RAD) or with p-Histone antibody H3 Ser-10 (1 : 50) (sc-8656-R, Santa Cruz Biotechnology) for 50 min in 2% normal horse serum. Then rinsing in TRIS for minimum 40 min took place, incubation with chromogen DAB (Sigma, D5637-SG) for 0-3 min. Rinsing again in distilled water for 10 min, dehydration for 10 min in two ethyl alcohols, 96% and 100%, respectively, rinsing in xylene 2 × 10 min and sizing of Histokitt specimens (MaeFour 1025/500).
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!