PLP1 Antibody (OASA03576)

Catalog No: OASA03576 (Formerly GWB-2BBCF0)

Size:100UG

Product Info

• Predicted Species Reactivity: Bovine
• Product Format: Liquid. Phosphate buffered saline
• Clonality: Monoclonal
• Clone: plpc1
• Isotype: IgG2a
• Host: Mouse
• Application: Flow cytometry|Immunofluorescence|Immunohistochemistry-Frozen|Immunohistochemistry-Paraffin|Western blot
• Additional Information: Fusion Partners: Spleen cells from immunised BALB/c mice were fused with cells of the mouse SP2/0 myeloma cell line.
  Preparation: Purified IgG prepared by affinity chromatography on Protein A from tissue culture supernatant
  Preservative Stabilisers: 0.09% - Sodium Azide
  Approx Protein Conc: IgG concentration 1 mg/ml
• Reconstitution and Storage: -20°C
• Immunogen: Synthetic peptide GRGTKF corresponding to C terminal region of myelin proteolipid protein.
• Purification: Protein G Purified
• Predicted Homology Based on Immunogen Sequence: Cow
• Concentration: 1 mg/ml
• Specificity: MYELIN PROTEOLIPID PROTEIN
• Protocol Information: Citation: 1: Bradl M, Bauer J, Flügel A, Wekerle H, Lassmann H. Complementary contribution of CD4 and CD8 T lymphocytes to T-cell infiltration of the intact and thedegenerative spinal cord. Am J Pathol. 2005 May;166(5):1441-50. PubMed PMID:15855644; PubMed Central PMCID: PMC1606398.
  Species: Rat
  Experiment Name: 1. Staining Conditions and Characterization of the Degenerative or Intact CNS—Methods of Quantification2. Establishing Staining Conditions to Identify Rat CD4 and CD8 T Cells3. Studying T Cells within the Spinal Cord—Methods of Quantification
  Experiment Background: The central role of T cells in inflammatory reactions of the central nervous system (CNS) is well documented. However, there is little information about the few T cells found within the noninflamed CNS. In particular, the contribution of CD4+ and CD8+ T cells to the lymphocyte pool infiltrating the intact CNS, the location of these cells in CNS white and gray matter, and changes in the cellular composition of T-cell infiltrates coinciding with degeneration are primarily undefined. To address these points, Monika et al. studied T cells in the intact and degenerative rat spinal cord.
  Experimental Steps: 1. Staining Conditions and Characterization of the Degenerative or Intact CNS—Methods of Quantification: For immunohistochemical analyses, tissue sections were stained according to standard procedures.The following antibodies were used: ED1 (directed against lysosomes of rat macrophages and activated microglia cells; , Oxford, UK), OX-6 (directed against MHC class II molecules; ), rabbit anti-proteolipid protein ,mouse anti-myelin oligodendrocyte glycoprotein (kindly provided by C. Linington, Martinsried, Germany, and by S. Piddlesden, London, UK), mouse anti-2’,3’-cyclic nucleotide-3’-phosphodiesterase (Affinity, Nottingham, UK), and rabbit anti-neurofilament (Chemicon, Temecula, CA). Cell counts were done by using an ocular morphometric grid. Evidence for neuroaxonal degeneration was the presence of ED1+ microglia cells surrounding and engulfing axons. The number of these cells was determined separately for each rat in a 0.25-mm2 area within the lateral funiculus of one representative thoracic section. Evidence for myelin degeneration was the presence of oligodendrocytes accumulating myelin in their cell bodies and the presence of ED1+ activated microglia cells engulfing myelin debris.4 Evidence for degenerative activity in the CNS was the activation of microglia cells, as revealed by reactivity with ED1 and OX-6. For each animal, the numbers of ED1+ and OX-6+ microglia cells per spinal cord cross-section were counted. The resulting values were used to calculate the average number of ED1+ and OX-6+ cells per animal. The spinal cord was considered intact in the absence of microglia activation, neuroaxonal degeneration, myelin degeneration, and oligodendrocyte death.2. Establishing Staining Conditions to Identify Rat CD4 and CD8 T Cells: CD8-positive rat T cells can be clearly identified in paraffin embedded, PFA-fixed tissue sections: They react with anti-CD3 antibodies directed against the T-cell receptor complex, and with anti-CD8 antibodies. For double stainings of T cells for CD3 and CD8 proteins, paraffin sections were deparaffinized in xylol and transferred to 90% ethanol. Endogenous peroxidase was blocked by incubation in methanol with 0.02% H2O2 (30 minutes, room temperature). Sections were then transferred to distilled water via a 90%, 70%, and 50% ethanol series. For antigen retrieval, sections were placed in a plastic coplin jar filled with ethylenediamine tetraacetic acid buffer, pH 8.5, and incubated for 60 minutes in a household food steamer device (MultiGourmet FS 20; Braun, Kronberg/Taunus, Germany). Afterward, they were incubated with 10% fetal calf serum in 0.1 mol/L PBS containing rabbit anti-human CD3 (polyclonal, cross-reactive to rat CD3; Dakopatts, Glostrup, Denmark) and mouse anti-rat CD8 (monoclonal OX-8, ) antibodies (4C, overnight). The sections were then washed in PBS and incubated with a mixture of alkaline phosphatase-conjugated goat anti-mouse antibodies (Dianova, Hamburg, Germany) and biotinylated donkey anti-rabbit (Amersham Pharmacia Biotech, Uppsala, Sweden) in fetal calf serum/PBS with 3% normal human serum (1 hour, room temperature). In a last step, avidin peroxidase (1:100, Sigma, Vienna, Austria) was applied for 1 hour at room temperature. Next, the alkaline phosphatase label was visualized with Fast Blue B base (Sigma), and the avidin peroxidase with 3,3’ diaminobenzidine-tetrahydrochloride (DAB, Sigma). With this staining procedure, Monika et al. were able to identify CD3+8+ T cells based on a dark violet staining, and CD3+8- T cells based on brown staining. To determine whether the CD3+8- T cells were CD4+ T lymphocytes, Monika et al. had to use cryosections, because anti-rat CD4 antibodies do not work on paraffin-embedded material. They performed triple labeling of spinal cord cryosections derived from a Lewis rat with CD4+ T-cell-mediated CNS inflammation. The sections were pretreated for 10 minutes with acetone. Then, W3/25 (against rat CD4, ) was diluted 1:50 in diluent (DAKO, Glostrup, Denmark) and applied (4C, overnight). The slides were washed with PBS, and incubated with a biotinylated sheep anti-mouse antibody (Amersham Pharmacia Biotech), diluted 1:200 in diluent (4 to 6 hours, room temperature). Afterward, the slides were washed and then incubated with streptavidin-Cy3 (Jackson ImmunoResearch Laboratories, West Grove, PA), diluted 1:75 in diluent containing 10% normal mouse serum (4C, overnight). Next, slides were washed with PBS and incubated overnight at 4C with fluorescein isothiocyanate (FITC)-labeled OX-8 (against rat CD8, ) and rabbit anti-human CD3 (cross-reactive with rat CD3, DAKO). Both antibodies were diluted 1:50 in diluent. Double labeling was finished by application of goat anti-rabbit Cy5 (Jackson ImmunoResearch) diluted 1:200 for 1 hour at room temperature. Slides were mounted with PBS/glycerol (1:9) containing 3% DABCO (Sigma) and inspected with a laser confocal microscope (LSM-410; Carl Zeiss, Jena, Germany). Excitation wavelengths of 488 nm (for FITC-labeled CD8), 543 nm (for Cy3-labeled CD4), and 633 nm (for Cy5-labeled CD3) were used to generate fluorescence emission, which was digitally translated to green (FITC), red (Cy3), and blue (Cy5) signals. With this staining protocol, we were able to verify that CD3+8+ T cells were CD4-, and CD3+8- T cells were CD4+.3. Studying T Cells within the Spinal Cord—Methods of Quantification: At least 10 spinal cord sections per adult animal were evaluated to determine the location of CD4+ and CD8+ T cells within the spinal cord, and to calculate the average number of CD4+ and CD8+ T cells per spinal cord section and animal. For all quantitative evaluations, between 4 and 10 animals per age group and genotype were analyzed. Quantification of T cells was done on complete spinal cord sections. Occasionally, hemisections were included. Because Monika et al. wanted to further validate the counts based on immunohistochemical stainings of tissue sections, they made an additional experiment in which they isolated T cells from the spinal cords of aged PLP transgenic rats, and studied these cells by cytofluorometry. As controls, T cells derived from the peripheral blood of the same animals were used. For staining, the cells were first incubated with CD4-FITC or CD8-FITC, diluted 1:100 in PAB (1 x PBS containing 2% fetal calf serum and 0.01% sodium azide) (30 minutes, 4C). Next, the cells were washed in PAB and reacted with R73 (specific for the rat T-cell receptor, ) or isotype-matched mouse IgG (negative control), both diluted 1:100 in PAB (30 minutes, 4C). Afterwards, T cells were washed and incubated with RPE-Cy-5-labeled secondary anti-mouse antibody (DAKO) (30 minutes, 4C). Finally, T cells were washed and cytofluorometrically analyzed. The percentage of R73+CD4+ or R73+CD8+ T cells in the R73+ T-cell population was determined with Lysis II software (Becton Dickinson, Heidelberg, Germany).
  Other Reagents Used: Paraformaldehyde (PFA)
  Number Of Protocols: 3
• Reference: 1. Rosetti, C.M. & Maggio, B. (2007) Protein-induced surface structuring in myelin membrane monolayers. Biophys J. 93: 4254-67.
  2. Grade, S. et al. (2010) Functional identification of neural stem cell-derived oligodendrocytes by means of calcium transients elicited by thrombin. Rejuvenation Res.13: 27-37.
  

Target Info

• Gene Symbol: PLP1
• Alias Symbols: PLP
• NCBI Gene Id: 281410
• Protein Name: Myelin proteolipid protein
• Description of Target: MOUSE ANTI MYELIN PROTEOLIPID PROTEIN
  
• Uniprot ID: P04116
• Protein Accession #: NP_776574.2
• Protein Size (# AA): 277