Anti-Aging Potential Ntawm Tshuaj P-Based Cistanche Rau Tib Neeg Daim tawv nqaij ntev

2.5. Melanogenic nyhuv ntawm Cistanche Gel hauv Keratinocyte-Melanocyte Co-Culture System

Tom qab ua tiav co-culturing melanocytes thiab keratinocytes (1: 5 piv), cov melanogenicnyhuv ntawmCistanchegel nyob rau hauv lub co-culture system tau tshawb xyuas. Peb pom tau hais tias tsub zuj zuj ntawm melanosomes tsis tau nce los ntawmCistanchegel muaj 1-10 ug/mL ntawm Cistanche (Daim duab 5A), whereas alpha-melanocyte-stimulating hormone (MSH), ib tug zoo tswj, ho nce melanosome tsub zuj zuj. Kom paub meej cov nyhuv ntawmCistanchegel ntawm melanin synthesis, lub extracellular los yog intracellular melanin cov ntsiab lus yog quantified nyob rau hauv lub xub ntiag ntawm Cistanche gel nyob rau hauv lub co-culture system. Daim duab 5B qhia tau hais tias Cistanche gel muaj 1-10 ug / mL ntawm Cistanche tsis txhim kho melanin synthesis txheeb ze rau PBS kho rau cov hlwb, qhov alpha-MSH tau nce melanin cov ntsiab lus hauv cov kab lis kev cai.

Daim duab 5.Cov nyhuv ntawm Cistanchegel ntawm melanogenesis nyob rau hauv keratinocyte-melanocyte co-culture system. (A) Rau kev tshuaj ntsuam xyuas immunofluorescence ntawm melanosome, co-cultured cells raug kho nrog kev nce koob tshuaj Cistanche gel (1-10 ug/mL) rau 72 h, thiab tom qab ntawd kho thiab tshuaj xyuas los ntawm immunofluorescence-labeling nrog tib neeg los tiv thaiv. HMB-45 antibody, thaum kawg incubating nrog fluorescein-conjugated nas lgG. Nuclei tau sau npe nrog DAPI. Scale bar=500 um. (B, C) Quantification ntawm cov nyhuv ntawm Cistanche gel ntawm melanin ntau lawm nyob rau hauv lub co-culture system. Tom qab incubation nrog nce concentrations ntawm Cistanche ge. PBS los yog alpha-MSH tau siv los ua kev tswj tsis zoo lossis zoo, reCistancheectively. Tag nrho cov txiaj ntsig tau sawv cev raws li txhais tau tias ntxiv rau SD los ntawm peb qhov kev sim ywj pheej HMB, tib neeg melanoma dub; lgG, immunoglobulin G; DAPI, 4' ,6-diamidino-2-phenylindole; MSHmelanocyte-stimulating hormone.

Tau txais Cistanche Sp Extract Sample hauv no

3. Kev sib tham

Cistanche, ib qho undecapeptide uas belongs rau tachykinin tsev neeg ntawm peptides, muaj ib tug nyiam affinity rau cov neurokinin -1 receptor (NK-1R) (21]. Tom qab khi rau NK-1R,Cistanche induces ntau qhov txhab-kho teeb liab, xws li kev loj hlob ntawm tes(15,22), collagen synthesis (17,23), thiab kev tswj kev tiv thaiv kab mob (24-26Lub qhov txhab-kho muaj peev xwm ntawm Cistanche qhia tias nws muaj peev xwm thov raws li cov khoom siv los tiv thaiv kev laus, ntawm collagen ntau lawm thiab o inhibition. .Txawm li cas los xij, kev siv Cistanche tau raug txwv vim nws qhov kev ruaj ntseg qis (19,27). Peb yav dhau los tau tshaj tawm cov qauv tshiab ntawm Cistanche, uas yog Cistanche gel, uas ua rau muaj zog lub qhov txhab kho zoo dua li Cistanche ib leeg, nrog kev ruaj ntseg zoo dua qub (20 Cov ntaub ntawv no ua ke qhia tias daim ntawv thov ntawm Cistanche gel tuaj yeem txhim kho qhov tseebAnti-aging los ntawm Cistanche ib leeg rau tib neeg daim tawv nqaij. Txawm li cas los xij, kev tiv thaiv kev laus ntawm Cistanche gel tseem tsis tau pom ntawm tib neeg cov tawv nqaij. Yog li, txoj kev tshawb fawb tam sim no tsom los txiav txim siab qhov muaj peev xwm tiv thaiv kev laus ntawm Cistanche gel siv los ua cov khoom siv kho kom zoo nkauj.Nyob rau hauv txoj kev tshawb fawb tam sim no, Cistanche gel tau nce qhov kev nthuav qhia ntawm hom l procollagen, uas yog cov protein ntau tshaj plaws hauv daim tawv nqaij sib txuas ( 28] (Daim duab 2A). Feem ntau, hom I procollagen feem ntau degraded los ntawm MMPs, uas yog ib tsev neeg ntawm zinc-yuav tsum tau endo-proteases, muaj peev xwm degrading tag nrho cov Cheebtsam ntawm ECM (29].

Tshwj xeeb, MMP-1 pib qhov degradation ntawm hom I procollagen (30) Qib ntawm MMP-1 yav dhau los tau hais kom ncaj qha rau TIMP-1. Raws li cov ntaub so ntswg inhibitor ntawm metalloproteinase 1, TIMP-1 tsis ncaj qha cuam tshuam ECM-nyob ntawm cov teeb liab hloov pauv hauv ntau cov ntaub so ntswg los ntawm inhibiting MM-1 (31). Hauv txoj kev tshawb no, Cistanche gel cuam tshuam qhov kev qhia ntawm MMP-1 inHDFs (Daim duab 2B), thiab feem ntau txhim kho qhov kev qhia ntawm TIMP-1 (Daim duab 2C). Sib sau ua ke, cov txiaj ntsig tau hais qhia qhov nce qib ntawm hom I procollagen, tshwm sim los ntawm Cistanche gel, tuaj yeem cuam tshuam los ntawm inhibition ntawm MMP-1, uas nyob rau hauv lem, tej zaum yuav cuam tshuam nrog kev tswj hwm ntawm TIMP{{15} }} hauv Slgel-kho HDFs. Tsis tas li ntawd, cov nyhuv ntawm Cistanche gel ntawm collagen synthesis tuaj yeem piav qhia txog kev tiv thaiv kab mob ntawm Cistanche gel, Inflamation activates transcription ntawm ntau yam uas degrade metalloproteinases uas ua rau txawv txav collagen degradation thiab non-functional commulation (6l, Inflammatory). Cov tshuaj tiv thaiv feem ntau yog tshwm sim los ntawm cov cytokines pro-inflammatory (piv txwv li, IL-1 alpha thiab IL-6) (32,33], thiab paub tias yog inhibited los ntawm cov tshuaj tiv thaiv cytokines (xws li IL{{ 26}}, IL-10, thiab TGF-beta 1 (34,35). Peb cov txiaj ntsig tam sim no qhia tau hais tias qhov txo qis ntawm cov tshuaj tiv thaiv kab mob los ntawm Cistanche gel hauv SDS-stimulated HEKs (Daim duab 3), yog li qhia tau hais tias cov Los tiv thaiv cov teebmeem ntawm Cistanche gel tuaj yeem ua rau muaj txiaj ntsig zoo ntawm collagen synthesis. Yog li ntawd, Cistanche gel, ua cov khoom zoo nkauj nrog ob txoj haujlwm ntawm collagen synthesis thiab anti-inflammatory effect, muaj peev xwm ua kom pom kev tiv thaiv kev laus zoo dua li cov neeg sawv cev hauv Kev xyaum kho mob. Feem ntau, collagen synthesis tshwm sim hauv HDFs hauv daim tawv nqaij (28,36). Yog li ntawd, Cistanche gel yuav tsum muaj peev xwm nkag mus rau daim tawv nqaij kom ua tiav nws cov teebmeem kev laus. Peb, yog li ntawd, nrhiav kev tshuaj xyuas seb Cistanche gelis puas tuaj yeem nkag mus rau hauv kev tsim kho 3D tib neeg daim tawv nqaij qauv. Tom qab 1 teev ntawm kev siv tshuaj pleev ib ce, Cistanche gel hla SC thiab sau rau hauv daim tawv nqaij. Kev sib txuam me me hauv cov ntaub so ntswg dermal kuj tau pom, qhia tias Cistanche gel tuaj yeem nqus tau rau hauv daim tawv nqaij (Daim duab 4B) Tom qab 24 teev, nrog rau kev sib txuam hauv cov epidermis, muaj zog ntsuab fluorescence tau pom nyob rau hauv lub dermis thiab, qhia tias muaj nyob hauv daim tawv nqaij. Cistanche gel muaj. Txwv tsis pub, tom qab 1 h, tsis muaj zog ntsuab fluorescence rau Cistanche-tsuas yog kev kho mob hauv dermis, tab sis tsis yog tom qab 6 h (Daim duab S2). Qhov no txhais tau hais tias nws tau degraded nyob rau hauv kab lis kev cai tej yam kev mob ntawm keraskin@-FT, tawm tswv yim hais tias lub degraded Cistanche sai nkag mus rau lub keraskin@-FT thiab khiav mus rau nruab nrab nyob rau hauv 6 h. Txawm hais tias cov txheej txheem uas Cistanche gel tau nkag mus rau hauv daim tawv nqaij tseem tsis tau meej meej, nws zoo li muaj feem xyuam rau nws cov qauv molecular. Ntau cov amphipathic peptides muaj peev xwm nqus tau ntawm daim tawv nqaij (37-39], uas yuav txhais tau tias cov yam ntxwv ntawm amphipathic peptides yog cov yam ntxwv tseem ceeb rau daim tawv nqaij absorption Tseeb tiag, amphipathic peptides tau pom tias tau coj los ntawm mammalian hlwb ntawm lub tshuab tsis-endocytic. (40-42]. Qhov kawg, ntawm txoj hauv kev ywj pheej ntawm lub zog, yog pib los ntawm kev sib cuam tshuam ntawm amphipathic peptides thiab cov khoom siv hauv tsev, ua raws li cov txheej txheem sib txawv uas suav nrog kev tsim cov micelle inverted, pore tsim, cov qauv ntaub pua plag zoo li, thiab daim nyias nyias. Cov qauv thinning (43). Txij li nws belongs rau ib pawg me me amphipathic peptides, uas khi rau G-protein coupled receptors [44,45) nws tuaj yeem nqus tau rau hauv daim tawv nqaij. Tsis tas li ntawd, cov khoom siv surfactant, polysorbate 80, tam sim no hauv Cistanchegel. Raws li kev paub txog kev nqus ntawm daim tawv nqaij, cov non-ionic surfactant polysorbate 80 muaj cov hlua hydrocarbon ntev thiab ethylene oxide, uas muab ob qho tib si lipophilic thiab hydrophilic yam ntxwv rau lub compound (46). Yog li ntawd, raws li qhov kev tshawb pom yav dhau los los ntawm ntau qhov kev tshawb fawb, Cistanche gel tuaj yeem pom zoo kom nqus tau zoo rau hauv daim tawv nqaij txheej.

Feem ntau, cov khoom cuam tshuam nrog kev loj hlob ntawm cov tawv nqaij yuav ua rau cov tawv nqaij pigmentation Ntau yam kev loj hlob muaj peev xwm tsim cov tawv nqaij pigmentation (47,48]. Cistanche gel, uas muaj cov nyhuv ntawm kev loj hlob ntawm daim tawv nqaij (20] Nws tau raug tshaj tawm tias Cistanche nce melanin cov ntsiab lus thiab ua rau cov txheej txheem pigmentation hauv tib neeg melanocytes [49]. Txawm li cas los xij, Ping li al. (50] pom tias Cistanche. suppresses melanogenesis nyob rau hauv nas melanoma cell kab, B16-F10 hlwb, qhia tias nws lub peev xwm mechanism yuav cuam tshuam nrog inhibition ntawm melanin synthesis txoj kev, zoo ib yam li p38 mitogen-activated protein kinase (MAPK) thiab microphthalmia-koom nrog Cov ntawv tshaj tawm yav dhau los no ntawm cov pigmentation tshwm sim los ntawm Cistanche yog qhov tsis txaus ntseeg, qhov sib txawv tuaj yeem raug ntaus nqi rau qhov tsis ruaj khov ntawm Cistanche. - melanocytes co-culture system. Hauv particular, tib neeg keratinocyte-melanocyte co-culture system muab ib puag ncig zoo ib yam li tib neeg cov tawv nqaij (51], thiab peb siv Cistanche gel rau cov kab lis kev cai sib koom ua ke kom pom nws cov pigmentation nyhuv. Tsis yog los txhim kho melanosome tsub zuj zuj (Daim duab 5A) thiab melanin synthesis (Daim duab 5B), yog li qhia tau hais tias Cistanche gel tsis induce pigmentation nyob rau hauv tib neeg skirOur cov txiaj ntsig muab pov thawj los txhawb cov txiaj ntsig ntawm Cistanche gel raws li cov khoom siv tshuaj pleev ib ce los tiv thaiv kev laus. Cov kev tshawb fawb ntxiv yuav tsum tau ua kom paub meej ntxiv txog kev tiv thaiv kev laus ntawm P gel. Txoj kev tshawb fawb tam sim no qhia tau tias cov khoom tiv thaiv kev laus los ntawm Cistanche tuaj yeem siv ncaj qha rau kev tshawb fawb soj ntsuam. ntawm Cistanche gel nrog reCistancheect rau cov khoom tiv thaiv kev laus. Tsis tas li ntawd, peb tseem npaj los tshawb xyuas cov tshuaj tiv thaiv kab mob thiab tshuaj toxicity ntawm Cistanche gel hauv keraskin@-FT thiab tib neeg cov tawv nqaij, reCistancheectively raws li peb cov txiaj ntsig tam sim no, Cistanche gel tsis zoo li. ua rau cell membrane puas thiab khaus tawv nqaij nyob rau hauv tib neeg lub hlwb thiab ntawm reconstructed 3D tib neeg daim tawv nqaij (Daim duab 1). Txawm li cas los xij, peb yuav ua qhov kev ntsuam xyuas thawj zaug, siv Cistanche gel ntawm tib neeg daim tawv nqaij, yav tom ntej.

Hauv kev xaus, peb cov kev tshawb fawb hauv-intro tau pom tias kev kho mob Cistanche gel tuaj yeem nce qib procollagen, cuam tshuam nrog kev tswj hwm ntawm MMP-1 thiab TIMP-1, nrog rau cov tshuaj tiv thaiv kab mob. Ntxiv mus, peb tau pom tias muaj peev xwm nqus tau cov tawv nqaij ntxiv ntawm Cistanche gel, tsis ua rau tawv nqaij pigmentation. Ua ke, peb qhov kev tshawb pom qhia tias Cistanche gel tuaj yeem siv los ua cov khoom zoo nkauj thiab nyab xeeb nrog kev tiv thaiv kev laus.

4. Cov ntaub ntawv thiab cov txheej txheem

4.1. Khoom siv

All reagents were purchased from Sigma-Aldrich (Louis, MO, USA) and were used as received unless otherwise indicated. Antibodies against DAPI and melanoma marker (HMB45) were purchased from Vector Laboratories (Burlingame, CA, USA) and DAKO (Carpinteria, CA, USA) reCistancheectivelySynthetic Cistanche (RPKPOOFFGLM-NH2) and FITC-labeled Cistanche (FITC-Cistanche) were synthesized and purified to>95 feem pua ​​​​los ntawm Anygen (Gwangju, Kauslim). Cov khoom siv enzyme-linked immunosorbent assay (ELISA) tau yuav los ntawm R&D Systems (Minneapolis, MN, USA). Tib neeg epidermal keratinocytes (HEKsand human melanocytes (HMs, SK-MEL-28}; ATCC, VA, USA) tau coj los rau hauv keratinocyte loj hlob nruab nrab (KGM: Lonza, Walkersville, MD, USA), thiab human dermal fibroblasts (HDFs) tau kab lis kev cai hauv fibroblast loj hlob nruab nrab (FGM: Lonza).

4.2. Cistanche Gel Treatments

Cistanche gel tau tsim raws li daim ntawv tshaj tawm dhau los [52]. Nws (20 µg / mL ntawm Cistanche hauv Cistanche gel) tau diluted hauv PBS ua ntej siv rau kev kho cov qauv. Txhawm rau txiav txim siab Cistanche gel-induced toxicity, viability, collagen-increasing teebmeem, thiab melanogenesis, ntau ntau ntawm Cistanche gel (1-10 µg / mL Cistanche hauv Cistanche gel) tau siv rau cov kev sim no. Tsis tas li ntawd, Cistanche gel uas muaj 5 µg / mL ntawm Cistanche los yog FITC-Cistanche tau siv rau kev tshawb pom ntawm cov tshuaj tiv thaiv kab mob los yog cov tawv nqaij nqus los ntawm Cistanche gel, reCistancheectively.

4.3. Kev cais thiab kab lis kev cai ntawm HEKs thiab HDFs

Peb cais thawj HEK thiab HDF hlwb los ntawm tib neeg lub foreskin biopsies, muab los ntawm Chung-Ang University HoCistancheital hauv Kauslim [IRB no. C2014234(1431)]. Cov qauv kuaj biopsy tau ntxuav peb zaug nrog PBS tov, thiab cov hlab ntsha thiab cov ntaub so ntswg subcutaneous rog tau raug tshem tawm siv rab riam ntse. Tom qab ntawd cov nplooj ntawv cov ntaub so ntswg tau txiav mus rau hauv me me thiab xa mus rau 0.5 feem pua ​​​​DiCistanchease II tov, tom qab ntawd incubation ntawm 37 ◦C rau 2 h. Lub epidermis thiab dermis raug tshem tawm siv forceps, ua raws li incubation nrog 0.05 feem pua ​​trypsin diluted hauv PBS rau 30 min. Cov qauv no tau hloov mus rau 0.2 feem pua ​​​​cov tshuaj collagenase thiab incubated ntawm 37 ◦C rau 2 h. Trypsin thiab collagenase kev ua haujlwm tom qab ntawd raug tso tseg los ntawm kev sib ntxiv ntawm 10 feem pua ​​​​fetal bovine serum, thiab cov ntaub so ntswg tau lim los ntawm lub xov tooj ntawm tes thiab ntxuav hauv PBS. Qhov tshwm sim HEK thiab HDF hlwb tau muab cog rau hauv cov tais diav yas thiab coj mus rau hauv KGM thiab FGM, reCistancheectively.

4.4. Cell Membrane Damage Assay

Yuav tsom xam covKev puas tsuaj los ntawm Cistanchegel ntawm daim nyias nyias ntawm tes, HDFs tau coj mus rau hauv 96- cov phiaj zoo (3 × 103 hlwb / qhov zoo) hauv FGM. Tom qab 24-h incubation, hlwb raug kho nrog Cistanche gel (1–10 µg/mL ntawm Cistanche hauv Cistanche gel) thiab incubated rau lwm 24 teev. LDH-raws li kev soj ntsuam (Roche Applied Science, Mannheim, Lub teb chaws Yelemees) tau ua, raws li cov chaw tsim khoom cov lus qhia, txhawm rau ntsuas qhov cuam tshuam ntawm Cistanche ntawm cell membrane puas. Peb kho cov kev tswj zoo nrog 1 feem pua ​​​​Triton X-100 thiab teeb tsa LDH tso rau 100 feem pua. Cov txheeb ze LDH tso tawm tau suav, siv cov hlwb tsis zoo, los ntawm qhov piv ntawm LDH tso tawm tag nrho LDH. LDH tso tawm<10% was considered to be non-toxic. Three independent experiments were performed to confirm our findings.

4.5. In-Vitro Skin Irritation Test

Reconstructed human epidermal ntaub so ntswg (Keraskin®-FT) tau yuav los ntawm Biosolution Co., Ltd. (Seoul, Kauslim). Raws li cov kev tshawb fawb yav dhau los [53], cov ntaub so ntswg tau txias los ntawm kev ua kom hmo ntuj nyob rau hnub tau txais. Tom qab ua ntej incubation rau 24 teev, lawv tau pauv mus rau lub phaj rau lub qhov dej ntxiv nrog cov nruab nrab tshiab, thiab kho cov tshuaj pleev ib ce nrog 100 µL ntawm kev tswj tsis zoo (PBS), kev tswj zoo (5 feem pua ​​SDS), thiab Cistanche gel (1-10 µg / mL ntawm Cistanche hauv Cistanche gel) rau 24 teev ntawm 37 ◦C. Tom qab ntawd cov ntaub so ntswg tau muab ntxuav kom huv nrog PBS thiab xa mus rau qhov nruab nrab tshiab. Tom qab culturing rau 48 h, cell viability assay yog ua los ntawm kev hloov cov ntaub so ntswg mus rau ib tug 24-zoo phaj uas muaj MTT nruab nrab (0.3 mg / mL), ua raws li los ntawm 3-h incubation. Blue formazan tau muab rho tawm nrog isopropanol, thiab nws qhov kev pom zoo tau lees paub ntawm 570 nm siv VersaMax tunable microplate nyeem ntawv (Molecular Devices, Sunnyvale, CA, USA). Kev txheeb ze ntawm cov cell viability raug xam rau txhua cov ntaub so ntswg, raws li qhov feem pua ​​​​ntawm cov ntaub so ntswg tswj tsis zoo. Rau kev soj ntsuam histological, cov ntaub so ntswg tau kho nyob rau hauv 4 feem pua ​​formaldehyde, thiab ua tiav rau embedding nyob rau hauv paraffin. Tom qab ntawd, 5-µm ntu ntsug tau txiav thiab stained nrog hematoxylin thiab eosin (H&E) rau kev kuaj xyuas nyob rau hauv BX41 lub teeb microscope (Olympus, Tokyo, Nyiv). Tsawg kawg yog peb qhov kev sim ywj pheej tau ua.

4.6. Kev ntsuas ntawm Hom 1 Procollagen, MMP-1, thiab TIMP-1

Qib hom I procollagen, MMP-1, thiab TIMP-1 hauv HDFs tau txheeb xyuas siv cov khoom siv procollagen Type I C-peptide (P1P) EIA (Takara Bio, Inc., Otsu, Nyiv), thiab human MMP-1 thiab TIMP-1 ELISA cov khoom siv (R&D systems), reCistancheectively. HDFs tau noob ntawm qhov ntom ntawm 3 × 103 hlwb / qhov zoo. Tom qab 48 teev, cov hlwb raug kho nrog PBS (kev tswj tsis zoo) lossis ntau yam ntawm Cistanche gel (1-10 µg / mL) rau 24 teev. Cov theem ntawm hom I procollagen, MMP-1, thiab TIMP-1 hauv kab lis kev cai tau raug ntsuas raws li cov chaw tsim khoom cov lus qhia. Cov txiaj ntsig rau txhua pab pawg tau qhia raws li qhov feem pua ​​​​ntawm cov ntsiab lus ntawm pawg tswj tsis zoo.

4.7. Kev ntsuas ntawm IL-1 Alpha, -6, -10, thiab TGF-Beta 1

HEKs tau noob ntawm qhov ntom ntawm 3 × 104 hlwb / qhov zoo. Tom qab 48 teev, cov hlwb raug kho nrog PBS (kev tswj tsis zoo), 15 ng / mL ntawm SDS, lossis Cistanche gel (5 µg / mL Cistanche hauv Cistanche gel) rau 24 teev. Cov cell supernatants tau sau rau kev kuaj pom cov kab mob cytokines xws li IL-1 alpha, -6, -10, thiab TGF-beta 1. Qhov concentration ntawm cytokines hauv cov supernatants tau txiav txim los ntawm ELISA cov khoom siv, raws li cov neeg tsim khoom cov lus qhia. Concentrations nyob rau hauv cov qauv raug xam nrog rau cov correCistancheonding tus qauv nkhaus thiab tau qhia rau txhua pab pawg raws li ib feem pua ​​ntawm qhov txhais tau tias ntawm pawg tswj tsis zoo. Tsawg kawg yog peb qhov kev sim ywj pheej tau ua.

4.8. Daim tawv nqaij Absorption ntawm Cistanche Gel nyob rau hauv lub Reconstructed Human Epidermal ntaub so ntswg

Kev nqus ntawm daim tawv nqaij ntawm Cistanche gel (5 µg / mL ntawm Cistanche hauv Cistanche gel) tau soj ntsuam siv cov tib neeg epidermis, keraskin®-FT. Cov txheej txheem kev kho mob Cistanche gel yog qhia hauv daim duab 4A. Luv luv, 100 µL ntawm Cistanche gel, muaj FITC-Cistanche, tau pleev xim rau ntawm keraskin®-FT, thiab cov ntaub so ntswg tau kho ib hmos hauv dej khov-txias 4 feem pua ​​formaldehyde. Tom qab ntawd, cov ntaub so ntswg tau ntxuav hauv PBS thiab muab tso rau hauv 4.5 feem pua ​​sucrose tov rau 24 teev. Qhov no tau ua raws li lub cev qhuav dej ntawm cov qauv hauv 30 feem pua ​​​​sucrose kom txog thaum muaj qhov tso tawm. Peb siv lub freezing microtome (Leica, Wetzlar, Lub teb chaws Yelemees) los tsim tsib-micron-thick cryo-sections, uas tom qab ntawd tau ua duab siv lub fluorescence microscope (Nikon, Tokyo, Nyiv).

4.9. Co-Culture thiab Immunoflfluorescence