Eight‐micron‐thick formalin‐fixed paraffin‐embedded (FFPE) tissue sections from the frontal cortex, temporal cortex and hippocampus were cut from the cases listed in Table 1 . Sections were deparaffinised in xylene and rehydrated using graded alcohols. Immunohistochemistry for all antibodies required pressure cooker pre‐treatment for 10 minutes in citrate buffer pH 6.0. Aβ immunohistochemistry also required formic acid pre‐treatment prior to pressure cooking. Endogenous peroxidase activity was blocked in 0.3% H2O2 in methanol for 10 minutes and non‐specific binding blocked with 10% dried milk solution. Tissue sections were incubated with primary antibodies; Aβ (1:100; Dako); AT8 (tau, 1:600; Thermo); Iba1 (microglial, 1:1000; Wako); CD68 (microglial, 1:100, Dako); CR3‐43 (microglial, 1:150, Dako); P2RY12 (microglial, 1:100; Sigma); Glial fibrillary acidic protein (GFAP) (astrocytic, 1:1000 Dako) for 1 h at RT, followed by biotinylated anti‐rabbit IgG (1:200; Dako) or biotinylated anti‐mouse IgG (1:200; Dako) for 30 minutes at RT and Avidin‐Biotin complex (30 minutes; Dako). Colour was developed with di‐aminobenzidine/H202 (30 ). Stained sections were digitised using a Leica SCN400F slide scanner.
P2ry12
Manufactured by Merck Group
Sourced in Sweden
P2RY12 is a receptor protein expressed on the surface of platelets. It plays a key role in the regulation of platelet activation and aggregation. The P2RY12 receptor is involved in the signaling pathways that mediate the effects of the platelet agonist adenosine diphosphate (ADP).
Automatically generated - may contain errors
Lab products found in correlation
Lsm 700, by Zeiss (2 mentions)
Biotinylated anti rabbit igg, by Agilent Technologies (1 mentions)
Biotinylated anti mouse igg, by Agilent Technologies (1 mentions)
Avidin biotin complex, by Agilent Technologies (1 mentions)
Scn400f slide scanner, by Leica camera (1 mentions)
Cr3 43, by Agilent Technologies (1 mentions)
Tmem119, by Abcam (1 mentions)
Cd163, by Bio-Rad (1 mentions)
Alexa fluor 647 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 and 555 conjugated secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse f ab 2 fragment, by Jackson ImmunoResearch (1 mentions)
Glial fibrillary acidic protein (gfap), by Agilent Technologies (1 mentions)
Clone at8, by Thermo Fisher Scientific (1 mentions)
Hydrogen peroxide, by Vector Laboratories (1 mentions)
Vectastain elite, by Vector Laboratories (1 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (1 mentions)
Iba 1, by Abcam (1 mentions)
Foxa2, by R&D Systems (1 mentions)
5 protocols using p2ry12
1
Immunohistochemical Analysis of Neurodegenerative Markers
2
Immunophenotyping of Donor-Derived Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To investigate expression of CAM markers in donor-derived cells of the LM and PV space, three patients with long survival after PBSCT and consecutively high numbers of engrafting donor-derived cells were chosen. In these patients, at least 100 perivascular and leptomeningeal Y+ cells pooled from cortical, hippocampal and cerebellar samples were assessed for Iba1 (Abcam, clone EPR 16588), CD206 (Abnova, clone 5C11) and Siglec1 expression. For microglia quantification, cortical samples were assessed for Iba1 (Abcam, clone EPR 16588), P2RY12 (Sigma-Aldrich, polyclonal), TMEM119 (Abcam, polyclonal) and GLUT5 (Sigma-Aldrich, polyclonal).
3
Immunohistochemical Analysis of FFPE Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Adjacent sections of 5 μm thickness from Formalin Fixed Paraffin Embedded (FFPE) samples were used for immunohistochemical analysis. The sections were incubated with antibodies against P2RY12 (1:100; Sigma, Sweden); CD68 (1:800; Dako, Denmark) GFAP (1:200; Dako, Denmark); CD45 (1:100; Dako, Denmark) and CD163 (1:400; Abd Serotec, USA). The staining procedure and scanning of the stained sections were performed according to the protocol described previously [40 (link)]. For double labeling, Alexa Fluor 488 and 555-conjugated secondary antibodies (1:200, Thermo Fisher Scientific, The Netherlands) were used for detection. For triple staining, Goat anti-Mouse F(ab)2 fragment (Jackson ImmunoResearch, WestGrove, PA, USA) was used to block background epitopes and Alexa Fluor 647-conjugated secondary antibody (Thermo Fisher Scientific, The Netherlands) was used for detection. The fluorescent labeled samples were analyzed by using the confocal microscope LSM 700 (Zeiss, The Netherlands). Signal positive areas and staining intensity were quantified using the Image J program to five high power field (40x) areas of each immunostained slide.
4
Immunohistochemical Analysis of Alzheimer's Pathology
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunohistochemistry was performed on 4 μm paraffin-embedded sections, targeting the markers of AD pathology pan-Aβ (clone 4G8, BioLegend), Aβ42 (clone 21F12, Elan Pharmaceuticals Ltd) and the phosphorylated (p) tau (clone AT8, ThermoScientific) protein, as well as the microglial motility-related proteins Iba1 (rabbit polyclonal, Wako Chemicals), CFL1 (polyclonal rabbit, ThermoScientific), CORO1A (rabbit polyclonal, LifeSpan Biosciences) and P2RY12 (rabbit polyclonal, Sigma Aldrich). Bound antibodies were visualized using the avidin–biotin–peroxidase complex method (Vectastain Elite, Vector Laboratories) with 3,3′-diaminobenzidine as chromogen and 0.05% hydrogen peroxide as substrate (Vector Laboratories). All sections were counterstained with haematoxylin, then dehydrated and mounted in Pertex (Histolab Products AB). The staining was performed in two batches with each batch containing cases from all groups (Control, AD, iAD). All experiments included a negative control slide incubated in buffer with no primary antibody and a positive control slide containing a specific tissue type known to express the protein of interest (e.g. tonsil).
5
Characterization of Pluripotent and Differentiated Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunocytochemistry was used to assess biomarker expression to characterize each of the cell types shown in this publication. Briefly, samples were fixed in PFA and permeabilized (0.03% triton when necessary) and blocked (BSA or serum) as required depending on the biomarker being analyzed on hESCs (OCT4 (DSHB), SSEA4 (DSHB)) or our differentiated cell cultures: dopaminergic neurons (TH (Pelfreeze), FOXA2 (R&D Systems)) or microglia (IBA1 (Abcam), P2RY12 (Sigma), CX3XR1 (Biolegend), PU.1 (Cell Signaling Tech)). Fluorochrome conjugated secondary antibodies were used to image our samples in an epifluorescence or confocal microscope. Alkaline phosphatase activity was measured using Vector® Black Substrate Kit, Alkaline Phosphatase (Vector Laboratories). Specific details on the protocol used can be found in protocols.io (https://doi.org/10.17504/protocols.io.yxmvm3146l3p/v1 ). For specific details on our staining of pluripotency markers in our hESCs consult: https://doi.org/10.17504/protocols.io.b4yyqxxw . OCT4, SSEA4 and AP Images from our hESC cultures were captured using a 10X objective on a fluorescence microscope (Zeiss ZEN 3.8).. Magnification may differ depending on which microscope-camera set was used to capture the images. This was a result of which team within the collaboration was in charge of generating a specific cell line and its analysis through the QC steps.
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!