For the best experience on the Abcam website please upgrade to a modern browser such as Google Chrome

Hello. We're improving abcam.com and we'd welcome your feedback.

Hello. We're improving abcam.com and we'd welcome your feedback.

Infomation icon

We haven't added this to the BETA yet

New BETA website

New BETA website

Hello. We're improving abcam.com and we'd welcome your feedback.

Take a look at our BETA site and see what we’ve done so far.

Switch on our new BETA site

Now available

Search and browse selected products

  • A selection of primary antibodies

Purchase these through your usual distributor

In the coming months

  • Additional product types
  • Supporting content
  • Sign in to your account
  • Purchase online
United States
Your country/region is currently set to:

If incorrect, please enter your country/region into the box below, to view site information related to your country/region.

Call (888) 77-ABCAM (22226) or contact us
Need help? Contact us

  • My account
  • Sign out
Sign in or Register with us

Welcome

Sign in or

Don't have an account?

Register with us
My basket
Quick order
Abcam homepage

  • Research Products
    By product type
    Primary antibodies
    Secondary antibodies
    ELISA and Matched Antibody Pair Kits
    Cell and tissue imaging tools
    Cellular and biochemical assays
    By product type
    Proteins and Peptides
    Proteomics tools
    Agonists, activators, antagonists and inhibitors
    Cell lines and Lysates
    Multiplex Assays
    By research area
    Cancer
    Cardiovascular
    Cell Biology
    Epigenetics
    Metabolism
    Developmental Biology
    By research area
    Immunology
    Microbiology
    Neuroscience
    Signal Transduction
    Stem Cells
  • Customized Products & Partnerships
    Customized Products & Partnerships

    Customized products and commercial partnerships to accelerate your diagnostic and therapeutic programs.

    Customized products

    Partner with us

  • Support
    Support hub

    Access advice and support for any research roadblock

    View support hub

    Protocols

    Your experiments laid out step by step

    View protocols

  • Events
    • Conference calendar
    • Cancer
    • Cardiovascular
    • Epigenetics & Nuclear signaling
    • Immunology
    • Neuroscience
    • Stem cells
    • Tradeshows
    • Scientific webinars
    Keep up to date with the latest events

    Full event breakdown with abstracts, speakers, registration and more

    View global event calendar

  • Pathways
    Cell signalling pathways

    View all pathways

    View all interactive pathways

Products by research area:
  • Cancer
  • Cardiovascular
  • Cell Biology
  • Epigenetics & Nuclear Signaling
  • Developmental Biology
  • Immunology
  • Metabolism
  • Microbiology
  • Neuroscience
  • Signal Transduction
  • Stem Cells

Primary Cultures for IHC – Viability Assays

By Dr. Jean-Christophe Rochet *

A. Preparation of primary mesencephalic cultures

  1. The mesencephalic neurons and glia are dissociated from neuronal tissue with trypsin (final concentration, 26 mg/ml in 0.9% [w/v] NaCl)
  2. Cells are plated on coverslips previously treated with poly-L-lysine (5 mg/ml).
  3. The media consists of DMEM, 10% (v/v) FBS, 10% (v/v) HS, penicillin (100 U/ml), and streptomycin (100 mg/ml).
  4. Four days later, the cells were treated for 48 hr with AraC (20 mM) to inhibit the growth of glial cells.

B. Lentiviral transductions of primary cultures

  1. AraC-treated primary cultures are transduced with lentiviral particles in the presence of polybrene (6 mg/ml).
  2. Control cells were incubated without lentivirus.
  3. After a 72 hr transduction period, the cells were treated with fresh media for an additional 48 hr and analyzed immunocytochemically.

C. Immunocytochemistry of primary midbrain cultures

  1. Primary cells were fixed in 4% (w/v) paraformaldehyde in PBS for 30 min.
  2. The cells were permeabilized and blocked simultaneously for 1 hr with PBS containing 1% (w/v) BSA, 10% (v/v) FBS, and 0.3% (v/v) Triton X-100.
  3. After washing with PBS, the cells were treated overnight at 4°C with an anti-MAP2 monoclonal IgG (1:500) and an anti-TH polyclonal antibody (1:500) to monitor relative dopaminergic cell viability.
  4. To determine the lentiviral transduction efficiency, untransduced cells or cells transduced with lacZ lentivirus (expressing beta-galactosidase fused to the V5 epitope) are treated at room temperature for 1 hr with:
    • (i) an anti-V5 monoclonal antibody (1:200) and an anti-MAP2 polyclonal antibody (1:500), or
    • (ii) an anti-V5 monoclonal antibody (1:200) and an anti-TH polyclonal antibody (1:500).
  5. After washing, the cells were treated for 1 hr at room temperature with goat anti-mouse IgG conjugated to AlexaFluor® 488 (1:1000) and goat anti-rabbit IgG conjugated to AlexaFluor® 594 (1:2000).
  6. The primary and secondary antibodies were prepared by diluting stock antibody solutions in PBS with 1% (w/v) BSA.
  7. The coverslips were mounted onto slides using ProLong Gold Antifade® reagent, dried at room temperature overnight, and sealed with clear nail polish.

D. Measurement of primary neuron viability

  1. MAP2- and TH-immunoreactive primary neurons can be counted in 10 randomly chosen observation fields for each experimental condition at a total magnification of 200x.
  2. The data should be expressed as the percentage of MAP2-positive neurons that are also TH-positive.
  3. Each experiment should be repeated 3 to 4 times using embryonic neurons isolated from independent animals.
  4. Statistical analyses can be carried out using the program GraphPad Prism®, Version 4.0

Purdue University

Get resources and offers direct to your inbox Sign up
A-Z by research area
  • Cancer
  • Cardiovascular
  • Cell biology
  • Developmental biology
  • Epigenetics & Nuclear signaling
  • Immunology
  • Metabolism
  • Microbiology
  • Neuroscience
  • Signal transduction
  • Stem cells
A-Z by product type
  • Primary antibodies
  • Secondary antibodies
  • Biochemicals
  • Isotype controls
  • Flow cytometry multi-color selector
  • Kits
  • Loading controls
  • Lysates
  • Peptides
  • Proteins
  • Slides
  • Tags and cell markers
  • Tools & Reagents
Help & support
  • Support
  • Make an Inquiry
  • Protocols & troubleshooting
  • Placing an order
  • RabMAb products
  • Biochemical product FAQs
  • Training
  • Browse by Target
Company
  • Corporate site
  • Investor relations
  • Company news
  • Careers
  • About us
  • Blog
Events
  • Tradeshows
  • Conferences
International websites
  • abcam.cn
  • abcam.co.jp

Join with us

  • LinkedIn
  • facebook
  • Twitter
  • YouTube
  • Terms of sale
  • Website terms of use
  • Cookie policy
  • Privacy policy
  • Legal
  • Modern slavery statement
© 1998-2023 Abcam plc. All rights reserved.