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Lampiran 1.
Pembuatan pereaksi Wagner, Meyer, Oragend~rff dan Ninhidrin
I . Pereaksi Wagner. Sebanyak 2,5 g iodin dan 2 g kalium Mida dilarutkan dabm 200 rnC aqudes. Lamtan a b n berwama cokiat.
2. Pemksr' Meyer. Setengah gram kalium iodw d i m b a h dengan 1,36 gram raksa klorida dibnrtkan dalam 100 mL aqudes rnenghasifkan larutan yang tidak berwama.
3. Pereaksi Drsgendorff. Ke dalam hbu bl dimasukkan 0.8 g bismut nitrat kemudian ditambahkan 10 mC asam asetat glasial dan 40 rnL aquades. Ke dalarn labu k e 2 dibwt bNtan 8 g M u m iodida dalsm 20 mL aquades. Kedua hnrtan sebnjutnya d h p u r akan menghasilkan tanrtan yang berwarna merah lembayung.
4. Pereaksi Ninhidh. Lamtan ninhidrin dibuat dengan cam me4rutkan O , I g
hablur ninhidrin daQm 100 mL e t a d (96%) mengtrasilkan lanrtan yang tidak bemama.
Lampiran 2. Pembuatan Preparat Histoptologi 1) Penyiapan organ Isampling
Organ pankreas diarnbil secepat mungkin setelah tikus dibunuh. Organ dibersihkan dengan l a w n NaCl Mobgis (0,9 % ) dsn difiksasi dalam
hrutan Bouin -ma
24 Jam (Cardinal, Allan dan Cameron Cardinal, 1998).
2) PemotmganlTrimming
Organ dipotong degan ketebalan k 0,5 cm dan disown di dalarn cassete dan dimasukhn ke daiarn ktanjang khusus aulwnak b
epmssor.
3) Dehidrasi (AutomaticPllx;essor T i ,Sakurn RH 12 D )
Cassete dipindahkan dalam larutan bufier f M n 1O % (2x). Cassete dpindahkan dalam ke lamtan ethanol 80 %. Cassete dipindahkan dalam kelarutan ethanol 95 %.
Cassete dipindahkan dalam ke larutan ethanol absdut (3x1.
Cassete dipindahkan dalam ke Lnrtan xylol(2x). CaSSBte dipindabban d a m ke wax (paraplast).
4) Penarikan udara
Cassete dipindahkan ke dalam tangki hampa udara dan jaringan dikeluarkan dengan dat yang terdapat pada milipore. 5 ) Penanaman jaringan dalam paraplas IEmbedSng a y m n d e (pads suhu -
9°C). Setefah parafin dibekukan, cassete dan paraplas dikeluarkan dari
6 ) Sampel jaringan yang telah dipersiapkan diptong dengan rnikmtm dengan
ketebabn 3
-5
mikrometer. Sayatan yang didapatkan dilempelkan pada
objek glass.
7) Deparafinisasidan rehidrasi
Tafrapan deparafinasi dan rehidrasi s e m berikuf: Preparat dimasuttan ke dalam larutan xylol, 3 menit (2x). Preparat dimasukan Ice dalam alkohd a W u t , 3 mena (2x). ?reparat dimasukan ke dahm lamtan albhol90 %. 3 menit.
PFeparat d i m u k a n ke dabm lamtan alkofd 80 %, 3 menit.
Preparat di b i b dengan air mengalir, 1 menit.
Preprat dimasukan ke dalam IanRan hematoksilin, 6-7 menit. Preparat dibilas daQm air mengalir , 1 menit. Preparat dimasukkan ke dalarn bnrtan hematoksifin, Imend.
Preparat d i b i k dengan air mengah'r. Imenit. Pfeparat dimasukkan ke d a m bnrtan eosin. 1-5 menit. a
ereparat dibitas dengan air mengalif. 1 menit.
Preparat dimasukkan ke dalam alkohd 70 % , I 0 x dupan.
Preparat dimasuklcan ke dalarn alkohd 90 % , I 0 x ceiupan. r
Preparat dimasukkan ke dalam a J k M absolut. 10 x celupan. Preparat dimasukkan ke d a m afkohol abdtit, Imenk
Preparat dimasukkan ke dahm hrutan xylol3 menit (3x1. 10 ) Mounting
Preparat di beri satu tetes lanrtan DPX dengan menggunakan syringe b#. Pmparat ditutup dengan gelas penutup dan dimahah tidak tehenb.14~ gelembung udara. K e l e W n cairan di tepi gelas penutup difap dengan menggunakan tissue.
I.Persiapan Larutan a
Larutan Bouin dibual dari campuran asam pikrat-formafin 10% -
asam asetat gtasial dengan perbandingan 75:25:5mL Lanrtan m u m dikrornaf dengan asam suffaf : 0,15 g kalium dikromat, 0,15 mL asam sufat pekat d m 400 rnt aquakst. Lanrtan nafrium bisufi 5% : 20 g natrium bisuff~&lam 400 mL
aquadest.
Larutan hematoksilin : 0.5 g hematokiln, 50 m t aquajest, 50 mL k m alum, 2 mL M u m dikrwnat 5% clan 2 mL asam sum NIZ dicampur kemudian diinkubasi pada suhu W'C, selarna 48 jam.
Asam hrida f 46: 4 mL asam klorida dabm 400 mL quad& Phbxin : 0,5 g fluksin B dabm 100 mL quad& Asam phosphotungstrc 5 % : 20 g asam phosphotungstik dalam 400 mL.
2. Deparafinisasidan rehid&
Preparat difiksasi dalarn lanrtan Bouin, 12-24 jam.
Preparaf dibilas dengan air mengalif, 5 menit. Preparaf dimasukkan kedabm larutan W u m dikromat dengan asam sum, 5 menit Pernucatanwarna dengan larutan natrium b i s m 5 %, 3-5 menit.
Preparat dibiias dengan air mengalir , 5 menit.
Preparat dimasukasn ke dalam lanrtan hematosilin sampai sd beta berwama biru tua (30 menit).
Pernucatan wama dengan lanrtan asam IdoFidzt 1 % menit.
Preparat dibifas dengan air mengalir. sampai sel beta berwama biru jemih (5 menit).
Preparat dimasukkan kedalarn brutan phloxin, 30-45 menit. Pernucatan wama dengan lanrtan asam phosphotungstic 5 %, 1 menit. Preparat di bibs dengan air mengalir, sampai sel alpha rnenjadi merah (5menit).
Oiferensiasi pada alkoM95 %. Bib sel dpha tidak terwamai dengan baik maka dapat dlcuci dengan alkohol80 %, 15-20 defik.
Dehidrasi dan mounting.
tampiran 4. Kromatograrn ftaksi A-6 dan K 6 hasil analisis KCKT pada
k=24Q nm
Calculation Method: AREA%
Peak Quantltationr AREA
No.
RT
Area
Conc 1
BC.
1
1.73
16308745
100.000
BB
Pola krornatograrn A-6 basil elusi kbroform- metanol(9:l)
F c a i Cuanrltatlon:
ARER
Calculation Method: RREn%
No.
RT
Area
Concl
BC
1
1.76 2.73
35043626 G41f44 544760 1109246 56736
93.745
BV TBB
2 3
3.55
:
3.72
5 6
5.14 6.53 7.00
7
103993 1949
l.lB2 1.457 2.361 0.152 0.492 4.005
TBV TV3 3B
BB 83
Pola kromatogram K-5hasil elusi kloroform mumi
Lampiran 6. Kmnabgram fmksi A 4 dan K b has1 analisis KCKT pada A=220 nm
Peak Quantitation: ARE3 No .
L
Calculazlon Mcrhod: h 2 3 . 5
RT
Area
Conc 1
BC
1.73
17767349
100.000
BB
17767349
100.000
So.
i 2 3 1
KT
Area
Ccnc 1
BE
1.83
Z119fl638 107093 792888
BV
35945 9678
94.266 1.811 3.527 0.177 0.158 0.013
22483435
100.000
2.31
5
3.61 5.34 6.71
6
9.17
39693
rae TBB
BB BB
BB
Pda krPmatogram K-5 hasil dusi ldoroform murni
