OPALIN Antibody Protocol: IHC, WB & ICC Applications

OPALIN Antibody is a research-grade immunological reagent targeting the OPALIN (oligodendrocytic myelin paranodal and inner loop protein) antigen, formulated in PBS and suitable for applications including Western blotting, immunohistochemistry, and immunocytochemistry in studies of myelin biology and oligodendrocyte function.

Product SKU

Ab351131

Target Protein

OPALIN (Oligodendrocytic Myelin Paranodal and Inner Loop Protein)

Formulation

PBS only (no carrier protein or glycerol)

Recommended Applications

Western Blot (WB), Immunohistochemistry (IHC), Immunocytochemistry (ICC)

Reference / Source Code

absea#OC-036

Storage Buffer

PBS

Background: OPALIN and Its Role in Myelin Biology

OPALIN, also known as Oligodendrocytic Myelin Paranodal and Inner Loop Protein, is a transmembrane protein expressed specifically in oligodendrocytes of the central nervous system (CNS). It localizes to the inner tongue and paranodal loops of the myelin sheath, regions critical for axon–glia communication and myelin maintenance. Research into OPALIN is relevant to understanding demyelinating diseases such as multiple sclerosis (MS) and other CNS white-matter disorders. As a highly cell-type-specific marker, OPALIN antibodies are valuable tools for identifying mature oligodendrocytes and assessing myelin integrity in both in vitro and in vivo model systems.

Sample Preparation

For Western Blot (WB): Lyse cells or homogenize tissue (e.g., brain or spinal cord) in RIPA buffer supplemented with protease inhibitors. Centrifuge at 12,000 × g for 15 minutes at 4 °C and collect the supernatant. Determine protein concentration by BCA or Bradford assay. Load 20–40 µg of total protein per lane on an SDS-PAGE gel of appropriate percentage (typically 10–12% for OPALIN).

For Immunohistochemistry (IHC): Fix tissue in 10% neutral buffered formalin (NBF) for 24 hours, embed in paraffin, and section at 4–6 µm. Perform antigen retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) by heat-induced epitope retrieval (HIER). For frozen sections, fix in 4% paraformaldehyde (PFA) for 15–30 minutes at room temperature.

For Immunocytochemistry (ICC): Grow cells on coverslips or chamber slides to appropriate confluency. Fix with 4% PFA in PBS for 15 minutes at room temperature. Permeabilize with 0.1–0.3% Triton X-100 in PBS for 10 minutes if the target epitope is intracellular.

Antibody Incubation and Detection Protocol

Blocking: Block non-specific binding by incubating the membrane, section, or cells with 5% non-fat dry milk or 5% normal serum (species matched to secondary antibody host) in TBST or PBS-T for 1 hour at room temperature.

Primary Antibody Incubation: Dilute OPALIN Antibody (Ab351131) in blocking buffer at the manufacturer's recommended dilution. Incubate overnight at 4 °C with gentle agitation. Optimal working dilution should be determined empirically for each application and sample type.

Washing: Wash three times with TBST or PBS-T, each wash for 5–10 minutes with gentle agitation to remove unbound antibody.

Secondary Antibody and Detection: Apply an appropriate HRP-conjugated or fluorophore-conjugated secondary antibody directed against the primary antibody's host species. Incubate for 1 hour at room temperature. For WB, develop with ECL substrate and image. For IHC, apply DAB chromogen substrate, counterstain with hematoxylin, dehydrate, and mount. For ICC, apply anti-fade mounting medium containing DAPI for nuclear counterstaining and image by fluorescence microscopy.

Storage, Handling, and Troubleshooting Tips

Storage: Store OPALIN Antibody (Ab351131) at –20 °C for long-term storage. Avoid repeated freeze–thaw cycles; aliquot upon first use. The antibody is formulated in PBS only, without glycerol or carrier protein, so handle with care to prevent protein aggregation.

Troubleshooting:

• No signal: Verify antigen retrieval conditions for IHC; check primary antibody concentration and incubation conditions; confirm secondary antibody compatibility.
• High background: Increase blocking time or concentration; increase number and duration of wash steps; reduce primary or secondary antibody concentration.
• Non-specific bands (WB): Optimize gel percentage; confirm positive and negative control lysates; titrate antibody concentration.
• Weak signal: Extend primary antibody incubation time; optimize antigen retrieval; increase antibody concentration within recommended range.

Frequently asked questions

What buffer is OPALIN Antibody (Ab351131) formulated in?

OPALIN Antibody (Ab351131) is formulated in PBS only, without glycerol or carrier protein additives. This should be considered when aliquoting and storing the antibody to avoid protein aggregation or degradation.

Which experimental applications is OPALIN Antibody suitable for?

OPALIN Antibody (Ab351131) is recommended for Western blot (WB), immunohistochemistry (IHC), and immunocytochemistry (ICC) applications. Optimal dilutions for each application should be determined empirically by the end user.

What positive control samples are recommended for OPALIN Antibody?

Because OPALIN is specifically expressed in oligodendrocytes of the CNS, brain or spinal cord tissue lysates and sections from mammalian species are commonly used as positive controls. Cell lines with confirmed oligodendrocyte lineage may also serve as appropriate positive controls.

How should OPALIN Antibody be stored to maintain activity?

Store the antibody at –20 °C for long-term use. Aliquot into single-use volumes upon receipt to minimize freeze–thaw cycles, which can reduce antibody performance. Since the formulation is PBS only, avoid prolonged storage at 4 °C.

Why is antigen retrieval important for OPALIN IHC on FFPE sections?

Formalin fixation cross-links proteins and can mask epitopes, reducing antibody accessibility. Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) reverses these cross-links, restoring epitope availability and improving signal intensity for OPALIN detection in FFPE tissue sections.

References

1. Annexe: absea product reference OC-036. Supplier internal reference for OPALIN Antibody formulation details.
2. Kamholz J, et al. Identification and characterization of OPALIN, a transmembrane proteolipid expressed in the CNS. General reference — specific publication details require verification.

Products are supplied for research and development use only. Not for use in humans, animals, diagnosis, or therapy.