® IF555-Wheat Germ Agglutinin (WGA, Liab Teeb)

 

Khoom npe	Cat. Tsis muaj.	Spec.
iF555-Nplej Germ Agglutinin (WGA, Liab Teeb)	G1731	100 UL

 

 

Nplej kab mob agglutinin (WGA) yog ib qho lectin uas khi rau N-acetyl-D-glucosamine thiab sialic acid, thiab yog ib qho ntawm feem ntau kawm thiab siv cov chav kawm ntawm lectins. Tam sim no nws yog qhov feem ntau kawm thiab siv dav hauv chav kawm ntawm lectins.WGA khi rau glycoconjugates, thiab nws cov derivatives thiab conjugates tau dav siv rau kev sau cov cell membranes thiab fibrotic caws pliav rau cov duab fluorescence thiab tsom xam.Cov carbohydrate-binding tshwj xeeb ntawm WGA yog qhia rau ib ntu ntawm -1,4-GlcNAc-txuas residues, hu ua chitin dextrins. Txhua monosaccharide muaj ob qhov zoo ib yam, tsis muaj kev cuam tshuam los ntawm kev sib txuas ntawm qhov chaw sib txuas rau peb lossis plaub -1,4-GlcNAc units. Ntawm cov monosaccharides kuaj, tsuas yog GlcNAc khi rau WGA, ManNAc tsis ua, thiab GalNAc khi tsuas yog tsis muaj zog.WGA khi nrog siab affinity rau sab hauv GlcNAc residues loj oligosaccharides uas muaj Gal ({9}})GlcNAc (1-3) (piv txwv li, poly (aminolactose)-hom glycans) repeats.N-acetylneuraminic acid koom nrog hauv kev sib cuam tshuam qis ntawm WGA nkaus xwb. WGA qhia tau hais tias tus qauv nyuaj ntawm glycan tshwj xeeb uas tuaj yeem siv rau kev txheeb xyuas cov qauv ntawm cov carbohydrates nyuaj.Lub iFluor®555 affix ntawm WGA yog tej zaum lub brightest WGA affix. Nws nthuav tawm lub ci ntsa iab fluorescence ntawm iFluor® 555 dye. iFluor® 555 WGA affix khi rau sialic acid thiab N-acetylglucosaminyl residues li AF555 WGA coupler.

 

Nplej Germ Agglutinin (WGA) yog 36 kDa lectin feem ntau siv los sau cov kab mob mammalian, cell membranes ntawm Gram-zoo kab mob thiab cov poov xab, nrog rau cov pob txha thiab lub plawv sacral daim nyias nyias, thiab lwm yam, vim nws cov cuab yeej ntawm coupling rau N-acetyl. - -D-glucosaminyl residues thiab N-acetyl- -D-glucosaminyl oligomers hauv cell membranes. Thaum WGA siv rau cardiomyocyte staining, cell membranes ntawm cardiomyocytes tuaj yeem stained tawm kom seb lawv puas yog hypertrophic lossis tsis tuaj yeem pom los ntawm kev sib piv ntawm cov duab nrog cov pab pawg ib txwm muaj, thiab txoj kab uas hla thiab thaj tsam ntawm cov cell stained tuaj yeem pom. ntsuas nrog software tshwj xeeb, ua rau kev tsom xam seb cov cardiomyocytes puas yog hypertrophic lossis tsis.

 

 

Nkoj nrog dej khov; Khaws ntawm -20 degree rau 12 lub hlis.

 

 

Cheebtsam	G1722-50UL
iF555-Nplej Germ Agglutinin (WGA, Liab Teeb)	100 μL
Phau ntawv	1pc ib

 

 

1. Npaj koj tus kheej1 × PBS tsis muaj(pH 7.2-7.4, pom zooG4202), fixative muaj 3. 0-4 0% formaldehyde (pom zooG1101, muaj 4% PFA),Anti-fluorescence quenching sealer(pom zoo G1401), thiabnpaj-rau-siv DAPI staining tov(pom zooG1012).

2. Thaum siv cov khoom lag luam thawj zaug, centrifuge cov khoom tawg tag nrho ntawm qhov ceev ntawm 1 min kom tsis txhob poob ntawm cov kua raj phab ntsa. Nws raug nquahu kom faib cov khoom raws li tus nqi siv hauv ib qho kev sim kom tsis txhob poob ntawm cov kuab tshuaj los ntawm volatilization. Khaws ntawm -20 degree deb ntawm lub teeb.

3. Ua ntej kev sim ua ntej, aspirate 5 μL iF555-Nplej Germ Agglutinin (WGA, Liab Teeb) thiab sib tov nrog 1 mL PBS kom tauiF555-WGA ua haujlwm daws teeb meem. Cov nyhuv staining tej zaum yuav txawv rau ntau hom hlwb, thov xa mus rau cov ntaub ntawv rau qhov zoo tshaj plaws staining ua hauj lwm concentration los yog ua ntej-test kom paub.iF555-WGA ua haujlwm daws teeb meemyog npaj siv, khaws cia nyob rau hauv chav tsev kub thiab tiv thaiv los ntawm lub teeb, thiab siv nyob rau tib hnub.

 

 

Nyob ntawm tes staining (12-zoo phaj ua piv txwv)

1. Ntxuav cov hlwb nrog 1 × PBS tsis rau 2 zaug.

2. Ntxiv 100 μL ntawm iF555-WGA ua hauj lwm tov rau txhua lub qhov dej ntawm cov hlwb thiab incubate rau 10-30 min ntawm 37 degree, tiv thaiv los ntawm lub teeb.Nrhiav kev sib khi kom tiv thaiv cov kua evaporation. Tshem tawm cov tshuaj incubation thiab ntxuav nrog buffer tov 2-3 zaug rau 3-5 min txhua zaus.

3. Tom qab ua tiav lawm, cov hlwb raug ntxuav peb zaug nrog 1 × PBS buffer buffer rau 5 min txhua zaus.

4. Cov txiaj ntsig tau pom los ntawm fluorescence microscopy lossis laser confocal microscopy.

 

Kho cell staining (6-zoo phaj cell crawler ua piv txwv)

1

2. Ntxiv tus nqi tsim nyog ntawm fixative los npog lub hlwb thiab kho rau 10-30 min ntawm chav tsev kub.

3. Lub fixative tau aspirated thiab cov hlwb raug ntxuav nrog 1 × PBS tsis nyob rau hauv chav tsev kub 2-3 zaug rau 10 min txhua.

4. Tom qab lub cell crawler tau shaken me ntsis qhuav, kos ib lub voj voog nrog tus cwj mem histochemical kom cov hlwb nyob hauv nruab nrab ntawm lub voj voog. Noj 100 μL ntawm iF555-WGA ua hauj lwm tov los npog cov hlwb kom tiav thiab incubate ntawm 37 degree rau 30 min deb ntawm lub teeb. Ceev faj kom muab khi thiab tiv thaiv cov kua los ntawm evaporating kom qhuav cov slides.

5. Tom qab incubation tiav lawm, cov hlwb raug ntxuav peb zaug nrog 1 × PBS tsis rau 5 feeb txhua zaus.

6. (xaiv tau) DAPI staining tov khoom npaj tau muab ntxiv rau hauv cov crawler slides los npog cov hlwb, thiab cov staining nuclear tau ua rau 8 min.Lub hlwb raug ntxuav nrog 1 × PBS tsis muaj 2-3 zaug rau 30s -1 feeb txhua lub sijhawm.

7. Cov xovtooj ntawm tes tau shaken me ntsis kom qhuav thiab tig mus rau hauv ib lub swb nrog ib qho kev poob ntawm cov tshuaj tiv thaiv fluorescence quenching sealer, thiab cov sealer ntau dhau tau raug tshem tawm nrog daim ntawv phuam.

[Nco tseg]Kauj Ruam 6 thiab 7, tuaj yeem hloov tau nrog DAPI-muaj cov tshuaj tiv thaiv fluorescence quenching sealer (Cov Lus Pom Zoo G1407), uas ua tiav cov staining ntawm nuclei thiab sealing ntawm zaj duab xis tib lub sijhawm.

8. Cov txiaj ntsig tau pom los ntawm fluorescence microscopy lossis laser confocal microscopy nrog 555 (Ex/Em=556/574 nm) thiab DAPI (Ex/Em=364/454 nm) channels xaiv.

 

Staining ntawm cov ntaub so ntswg

1. Kev kho cov ntaub so ntswg ua ntej:

Frozen seem (cov ntaub so ntswg tshiab khov los yog cov ntaub so ntswg khov khov) tau tso tseg kom sawv ntawm chav sov li ob peb feeb thiab rov qab mus rau chav sov; cov seem tau submerged nyob rau hauv fixative rau 10-15 min ntawm chav tsev kub, tshem tawm thiab qhuav lawm, thiab ces moistened thiab ntxuav nyob rau hauv dej purified los yog 1 × PBS tsis rau tshem tawm cov residual fixative ntawm cov ntaub so ntswg.

Paraffin seem tau dewaxed thiab rehydrated;

Cov ntaub so ntswg (cov seem paraffin lossis cov seem khov ntawm cov ntaub so ntswg ruaj) tau muab tso rau hauv cov khoom siv kho mob uas muaj EDTA antigen kho tsis haum (pH 8.{1}}) hauv qhov cub microwave rau kho antigen. Nruab nrab cua sov rau 8 min nres hluav taws rau 8 min mus rau nruab nrab-tsawg tshav kub rau 7 min, cov txheej txheem no yuav tsum tiv thaiv ntau dhau evaporation ntawm tsis, tsis txhob qhuav cov slides. Tom qab qhov txias txias, cov slides tau muab tso rau hauv 1 × PBS tsis thiab ntxuav los ntawm co ntawm decolorizing shaker rau 3 zaug, txhua zaus rau 5 min.

2. Staining: Kos ib lub voj voog ncig cov ntaub so ntswg nrog tus cwj mem histochemical tom qab cov ntaub so ntswg qhuav me ntsis. Ntxiv 50-100 μL ntawm iF555-WGA ua hauj lwm tov rau lub voj voog kom npog cov ntaub so ntswg, thiab incubate ntawm 37 degree deb ntawm lub teeb rau 30 min. Saib xyuas kev sib khi kom tiv thaiv cov kua los ntawm evaporating thiab ziab cov slides.

3. Ntxuav: Tom qab ua tiav, cov qauv raug ntxuav peb zaug nrog 1 × PBS tsis pub rau 5 feeb txhua zaus.

4. Nucleus staining (optional): dropwise ntxiv npaj-rau-siv DAPI staining tov rau cov qauv los npog cov hlwb, thiab ua cov nucleus staining rau 8 min. Ntxuav cov qauv nrog 1 × PBS tsis 2-3 zaug rau 30 s -1 feeb txhua lub sijhawm.

5. Sealing: Tom qab cov seem tau shaken me ntsis qhuav, ntxiv ib qho poob ntawm Anti-fluorescence quenching sealer rau cov qauv thiab muab khi nrog cov ntaub npog tsim nyog.

[Nco tseg]Kauj Ruam 4 thiab 5, tuaj yeem hloov tau nrog DAPI-muaj cov tshuaj tiv thaiv fluorescence quenching sealer (Kev Pom Zoo G1407), uas ua tiav cov staining ntawm cov nucleus thiab sealing ntawm zaj duab xis tib lub sijhawm.

6. Microscopy: Fluorescence microscopy lossis laser confocal microscopy los soj ntsuam cov txiaj ntsig, xaiv 488 (Ex/Em=556/574 nm) thiab DAPI (Ex/Em=364/454 nm) channels.

 

 

1. Cov qauv uas tau kho rau lub sijhawm ntev (piv txwv li, paraffin seem) yuav tsum tau ua cov txheej txheem kho pyrolysis lossis lwm qhov txiaj ntsig zoo yuav tsis muaj zog lossis ze rau qhov tsis muaj.

2. WGA staining rau myocardial tsom xam yuav tsum muaj qib siab ntawm cov ntaub so ntswg myocardial, uas yuav tsum ua kom tiav thiab homogeneous, thiab nws yog qhov zoo tshaj plaws los muab cov qauv ntawm paraffin seem.

3. Rau cov qauv ntawm tes, tsis txhob siv cov tshuaj permeabilizing ua ntej staining, vim qhov no yuav ua rau staining ntawm cytoplasmic structural Cheebtsam.

4. Fluorescent dyes raug quenching, thiab nws raug nquahu kom kuaj thiab soj ntsuam cov duab kom sai li sai tau tom qab staining.

5. Thov hnav lub tsho (lab) thiab hnab looj tes thaum ua haujlwm.

 

Rau Kev Tshawb Fawb Siv Tsuas!

 

 

Cim npe nrov: ® if555-nqaij kab mob agglutinin (wga, lub teeb liab), Tuam Tshoj ® yog555-nqaij kab mob agglutinin (wga, lub teeb liab) manufacturers, lwm tus neeg, Hoobkas