31
.
v Argentaffin:
Cectione were stained with * nvnonlacal ' liver, in the dark fcr 3 hcorr at
6
C°C* Cricie were r i m e d with dis tilled veter and allowed to dry "before examination* Control: •tain* with dir tilled water*Ammonl&cal Cilver Jolution:
Ammonia £
8
C eolation it added dropwise to 1C Ag C x until the "brown precipitate first formed ir dissolved* Prerh1
C"' A£7C, ir added until the precipitate ;)urt begins; to reappear* The solution appeare op lercentand in dicrolved with
9
volumes of dir tilled water beforeu/e*
Chromaffin:
At for Ag method except hood*® Li chroma te rclution it used at a i t i n *
Control: fhtain* with C*2K Acetate buffer pH 4.1.
All etains- u»;ed for grid staining ia .a cytochemii try were centri fuged at
1
C,00
C r*p*m* for10
irdnutee before ute*b) Cytochemic&l localisation of Glycoproteins
i. Periodic cid - Chromic acid - liver Nethenamine (p^— Cr-Ag) - grid (Hambcurg et al*
1969
) Fix, Hint© etc* ac for kg methodUltrathin sections were supported on rt&i l o r e steel gride and *ctained* with the following solutions *
1
Periodic cid 2C minuter at rccm te per »ture Lit tilled water 3C "*5
r* "1C"" Chromic acid 5 " M " "
52.
I if tilled fcater t x 5 minuter. at rccm temper ature Lilver methenaoiine 40 minuter» in darky ut
6
C°C Pit.-tilled water Rapid r i m eControls £ tain with ilver I ethenamine only* filver kothenamine elation:
In a d rkrt cm, I ml* of * Ag are clowly added to 45 ml of
3
lethenociine (Jexsj&ine). white proceipitate ii initially formed but this diceolvee by rkaking.5
ml. of2
cdiam borite are finally added tc the solution*ii* leriodic cid - Bii.muth Gxynitrate (FA-71) - / rid (Ainsworth ot al* 1972).
Tiscue processing ar for P -Cr— g method
Ultrathin sections are s t i n e a with the following cclutionc at room temperature:
1
l eriodic acid10
minuterThorough rinre with distilled w ter Alkaline Bitmuth Cxynitrate 45 minutes Thorough rinre with dittilleu water
Control: leriodic reaction it blocked by incubting
r e c t ione with
1
& meta Amincphenol in glacial Acetic acid for 49 tai utec at room temperature* After r i m i n g withxetic acid sections are rinsed with dietilled water and then processed for I — Pi method*
Alkaline Firmuth Cxynitrate tolution:
40C g* of odium t - r t r a t e are dissolved in 1C ml. of 2k * aCTT* This tolution in added dxvpwire to £C0 ag of Bismuth oxynitrate with constant shaking. Initially the tolution ir cloudy but after the addition of t— £ al* it cleans* The stock tolutio it: ctored at 4CC and it diluted 1:50 before use*
33.
c) Cytochomical localisation of Proteoglycans
i. Bismuth nitrate pH 1.2 (Bi. 1.2) - {?rid ('jrith et al. 1967) Titruer were procemeci ar for the T -Cr-Ag methods. Ultras thin eectionc were me anted cn Nickel gride and tt-dnec. with the following solutions at roctr. tamper turo:
0.124 Nitric acid 5 e?inutec
0*5 Bismuth nitrate in 0.1K M t r i c acid 2C minutee C.
1
M Citric acid3 ^ 1
minuterThorough r i m e with di tilled water
Control: 1 tain with 0.1k l itric acid alone
ii. T a m i c acid - Ferric chloride (Tt-re) - grid (banner. et al. 1 9 7 0 Tiesuet were processed ae fcr the F .-Cr-Ag mathed.
1
ectiont were e t .ined with the following eolation? at room temperature:5 Tsniic acid
1
C minutesRapid rinse with di tilled w ter
? Ferric chloride
1
C mi iutee Rapid rinro with dirtilled water Control: Stain with Tannic acid only,iii. Colloidal Iron (Coll. Fe) — en bloc (Hayat 1 9 7 0
lor this; procedure it it nscerenry to prepare very small pieces of tissue cinco colloidal iron penetrates ticcuee- very poorly.
Tissues were fixed acccrdin to Fix. II. lube faet were then cut into
1
mm blocks, r i m e d with dittilled wuter and placed in colloidal iron solution for1
hour at rccs tecperature under continuous agitation.After r i m i n g ii C.2R Acetate buffer pH 1.7* ti ruef were dehydrated in th-nol nd processed for THK, o m i t i r g uranium
34.
Controls Fathylation "block - prior to en bloc rtaining
tireuer are incubated in acidified methanol ever ight ;t fcC°C. Colloidal Iren Solutions
Fee
1
2.75s B O1
C ml. Clycerol4
ml*2t; m m
2.2
&i.A
Ferric chloride ir completely dissolved in water by boiling for 5 minuter* Glycerol is added tc the filtered rolution and then ammonia added drcpwiee.
The final, ruit coloured eolation is dialjred through Vieking tubing ag ilart dir tilled w*ter for
72
heart* The dirtillod water ie changed 1C timer during this period. fter dialysis the colloidal ferric chloride solution increases; in volume to approximately?5
ml. The final pH of the solution is adjusted to 1*7 using glacial Acetic acid •iv* Colloidal Iron (Coll* Fe) - grid (Katuk'.g ct al. 1967} Tieruet were prepared a.*, for the P — Cr-Ag method
Ultrathin sections were mounted cn Uickel gridF and etained with the following solution;? at
rccra
temper ture:Colloidal Iron 1 hour
12
Acetic acid 3 x 9 minutesThorough rinse with distilled water
Cytocher ical localisation of cs.ticnt and -r <y microa.alysis i. Lanthanum Chloride (La) - en bloc (Veihe et al. 1977)
Lxci; ed ambul ieral tube feet were placed in an aerated ceo- ir ter bath at 4°C containing 10 ml. LaCl^.
After
45 minuter
tissues were rinradin sea water
aad processed t c / i rTSM
using Fix* II. Unst lined sections were examined.35.
ii* Aar. onium oxalate — en bloc (Caratgo & Fav&rd 1f>6£)
A* for L& method except t e vatcr bath cental nr 3 mi. Ammonium oxalate*
ill* Fctaceium lyroantiiBonate (K«*Ant) - en bloc
Initially the method of Bulger (l9t9) followed*
O /
Prefix* 1 hour, 0-4 C in 2*5 Glutaraldehyde/C*2K ThOFphate pH 7*6 + 0.21K nCl ♦ 2 1— pyroantimon-te.
H i m e * 9 mlnutec in ( *2H i horphv te p<i 7 mt + 0.2
1
K '.aCl ♦ 2 K-py roar, tissonat e •ubtequent proceeding ae for T' i , omitting uranium and lend etoining.
After nua»roua probleaw? with ui- to Ivin,.' I— pyroan t imon.t e in
phouph&te buffer alic poor rerulto, the method of Klein et *1* (