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Biological Description
| Specificity | Laminin Antibody (Mouse mAb) [E12M18] detects endogenous levels of total Laminin protein. |
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| Background | Laminins are heterotrimeric extracellular matrix glycoproteins assembled from α, β, and γ chains that associate through a coiled-coil domain into a cross-shaped molecule, with short arms mediating self-polymerization and a long arm bearing LG domains that engage cell-surface receptors. Laminin polymerization is initiated through interactions among the three short arms of separate trimers, driven primarily by the α-chain LN domain, generating a nascent two-dimensional lattice that becomes anchored to the cell surface. This cell-surface anchorage requires binding through the laminin LG domains to two principal receptor classes, integrins and dystroglycan, which act in a partially compensatory manner: either receptor alone can support laminin recruitment and basement membrane formation, but efficiency and downstream cell polarization are maximized when both receptors are engaged together, and loss of both abolishes laminin accumulation entirely and triggers apoptosis in affected cells. Once anchored, the nascent laminin scaffold recruits nidogen, type IV collagen, and heparan sulfate proteoglycans including perlecan and agrin, with nidogen forming a bridging link between laminin and the independently self-assembling type IV collagen network, and the two polymer systems together constituting the structural core of the mature basement membrane. Integrin and dystroglycan engagement additionally couples the assembled matrix to the underlying actin and keratin cytoskeletons, transmitting mechanical and signaling information bidirectionally across the cell surface and influencing cell shape, polarity, adhesion, and differentiation. Laminin isoform composition varies by tissue and developmental stage, with distinct α, β, and γ chain combinations defining basement membranes across epithelial, vascular, neural, and muscle tissue. Mutations affecting the α2 laminin chain or its dystroglycan and integrin receptor interactions disrupt sarcolemmal lattice organization in skeletal muscle and are a recognized cause of congenital muscular dystrophy. |
Usage Information
| Application | IHC, IF, ELISA | Dilution |
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| Reactivity | Feline, Human, Porcine | ||||
| Source | Mouse Monoclonal Antibody | MW | 337 kDa | ||
| Storage Buffer | PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3 | Storage (from the date of receipt) |
-20°C (avoid freeze-thaw cycles), 2 years | ||
| IHC |
Experimental Protocol：
Deparaffinization/Rehydration
1. Deparaffinize/hydrate sections:
2. Incubate sections in three washes of xylene for 5 min each.
3. Incubate sections in two washes of 100% ethanol for 10 min each.
4. Incubate sections in two washes of 95% ethanol for 10 min each.
5. Wash sections two times in dH2O for 5 min each.
6.Antigen retrieval: For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.
Staining
1. Wash sections in dH2O three times for 5 min each.
2. Incubate sections in 3% hydrogen peroxide for 10 min.
3. Wash sections in dH2O two times for 5 min each.
4. Wash sections in wash buffer for 5 min.
5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature.
6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C.
7. Remove antibody solution and wash sections with wash buffer three times for 5 min each.
8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature.
9. Wash sections three times with wash buffer for 5 min each.
10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use.
11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.
12. Immerse slides in dH2O.
13. If desired, counterstain sections with hematoxylin.
14. Wash sections in dH2O two times for 5 min each.
15. Dehydrate sections: Incubate sections in 95% ethanol two times for 10 sec each; Repeat in 100% ethanol, incubating sections two times for 10 sec each; Repeat in xylene, incubating sections two times for 10 sec each.
16. Mount sections with coverslips and mounting medium.
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| IF |
Experimental Protocol:
Sample Preparation
1. Adherent Cells: Place a clean, sterile coverslip in a culture dish. Once the cells grow to near confluence as a monolayer, remove the coverslip for further use.
2. Suspension Cells: Seed the cells onto a clean, sterile slide coated with poly-L-lysine.
3. Frozen Sections: Allow the slide to thaw at room temperature. Wash it with pure water or PBS for 2 times, 3 minutes each time.
4. Paraffin Sections: Deparaffinization and rehydration. Wash the slide with pure water or PBS for 3 times, 3 minutes each time. Then perform antigen retrieval.
Fixation
1. Fix the cell coverslips/spots or tissue sections at room temperature using a fixative such as 4% paraformaldehyde (4% PFA) for 10-15 minutes.
2. Wash the sample with PBS for 3 times, 3 minutes each time.
Permeabilization
1.Add a detergent such as 0.1–0.3% Triton X-100 to the sample and incubate at room temperature for 10–20 minutes.
(Note: This step is only required for intracellular antigens. For antigens expressed on the cell membrane, this step is unnecessary.)
Wash the sample with PBS for 3 times, 3 minutes each time.
Blocking
Add blocking solution and incubate at room temperature for at least 1 hour. (Common blocking solutions include: serum from the same source as the secondary antibody, BSA, or goat serum.)
Note: Ensure the sample remains moist during and after the blocking step to prevent drying, which can lead to high background.
Immunofluorescence Staining (Day 1)
1. Remove the blocking solution and add the diluted primary antibody.
2. Incubate the sample in a humidified chamber at 4°C overnight.
Immunofluorescence Staining (Day 2)
1. Remove the primary antibody and wash with PBST for 3 times, 5 minutes each time.
2. Add the diluted fluorescent secondary antibody and incubate in the dark at 4°C for 1–2 hours.
3. Remove the secondary antibody and wash with PBST for 3 times, 5 minutes each time.
4. Add diluted DAPI and incubate at room temperature in the dark for 5–10 minutes.
5. Wash with PBST for 3 times, 5 minutes each time.
Mounting
1. Mount the sample with an anti-fade mounting medium.
2. Allow the slide to dry at room temperature overnight in the dark.
3. Store the slide in a slide storage box at 4°C, protected from light.
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References
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Application Data
IHC
Validated by Selleck
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Immunohistochemical analysis of formalin fixed paraffin embedded human oral tumors tissue with F3833 at 1:25 dilution.