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Biologische Beschrijving
| Specificiteit | Loricrin C-terminal Antibody [D17A11] detecteert endogene niveaus van het C-terminale gebied van het totale Loricrin-eiwit. |
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| Achtergrond | Het C-terminale domein van Loricrin vormt de glutamine- en lysine-rijke ankerregio van dit dominante verhoornde omhulsel (CE) eiwit (goed voor meer dan 70% van de epidermale massa), met glycine-serine lussen met meerdere Q/K-paren – zoals Gln451/Lys454-clusters – die fungeren als primaire transglutaminase 1/3 (TGM1/3) substraten voor de vorming van ε-(γ-glutamyl)lysine isopeptidebindingen, verder versterkt door cysteïnedisulfidebruggen die onoplosbaarheid verlenen. Tijdens terminale differentiatie van keratinocyten medieert Ca²⁺-geactiveerd TGM1 sequentiële intra- en extracellulaire verknoping, waarbij C-terminale glutamineresten worden gedeamideerd tot glutamaten om lysine ε-aminogroepen voor nucleofiele aanval te primen, waardoor loricrin rigide wordt verankerd aan het involucrine/small proline-rich protein (SPR) scaffold in L-granulen en vervolgens aan het plasmamembraan, resulterend in een 10-20 nm dikke, mechanisch veerkrachtige schil die ondoordringbaar is voor water en pathogenen, met flexibele glycinelussen die trekspanning absorberen (modulus ~100 MPa). Deze post-translationele onoplosbaarheid via TGM-gekatalyseerde roosterpolymerisatie vestigt mechanistisch de competentie van de epidermale barrière, transcriptioneel aangedreven door NFAT, AP1 en NRF2-gemedieerde opregulatie van LOR. Dominante frameshift-mutaties die de C-terminus afkorten, produceren arginine-rijke nucleaire lokalisatiesignaal (NLS) peptiden die verkeerd lokaliseren naar de kern en de verknoping belemmeren, waardoor de terminale differentiatie dominant wordt verstoord en loricrin keratoderma (LK/Vohwinkel syndroom) ontstaat, gekenmerkt door palmoplantaire hyperkeratose, ichthyose en barrièrefaciliteit door incompetente verhoornde omhulsels. |
Gebruiksinformatie
| Toepassing | IHC, IF | Verdunning |
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| Reactiviteit | Rat, Human | ||||||
| Bron | Rabbit Monoclonal Antibody | MW | |||||
| Opslagbuffer | PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3 | Opslag (Vanaf de datum van ontvangst) |
-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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Referenties
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