Table of contents:

1. Fly rasing and breeding
2. crosses :
3. Eggs and embryos :
4. clones
5. Mutagenesis
6. stock collection
7. Genetic marker
8. Balancer
9. Injection of embryos
10.  Mikrosporidia
11. Präparations
12. Materials, equibment, etc

1. Fly rasing and breeding

The yield of fly vials - number of progeny - depends on various factors:
a) Number of females within the parent generation
b) Duration of egg laying (How many days have the parents been in the vial?)
c) Temperature (at 18°C the no of progeny is only 50% of no at 23°C, at 29ºC the no is even further reduced)
d) genetic constitution of the parents and the progeny
e) Quality of the food. A drop of fresh yeast significantly increases the yield. Soft food yields more progeny than hard food.

Estimates of the yield depending on no of females

size of food vial
 No of females
 period of egg lying (d)
 yield F1
big (55 mm)
 10 - 40
 3 - 5
 ca. 500 flies
middle (35 mm)
 5 - 20
 3 - 5
 ca. 150 flies
small (20 mm)
 1 - 10
 3 - 5
 ca. 60  flies
Minis
 1 - 3
 3 - 5
 ca. 20  flies


The number of males amoung the parents is not really important. Sufficient numbers are from 20% to 5x of the number of females. It is not required to remove the parents after a given period of egg laying, if the progeny can be unambiquously identified. It is however recommended to flip the flies into new vials and finally discard the parents. To obtain larger numbers of flies, a high number of parents (50-100 pairs) are filled into a big yeasted vial. The flies are flipped into new vials daily or every second day. To have a constant supply of virgin flies, it is better to keep lower numbers in a vial, which are flipped weekly.

A good yield is obtained only if the vials are regularly checked and kept in good shape.
  • Ideally the vials are moderately humid, the larvae leave the food and crawl up the wall as big 3rd instar for pupation.
  • dry food vials: the food retracts from the wall of the vial. The flies get stuck in the gaps, Larvae dry up and pupate within the food. To prevent this, set up the crosses with more parents. You may cut crosswise two slites into the food, to prevent retraction from the wall. For very dry vials you may add a few drops of water.
  • wet food: caused by overcrowding by too many larvae, if food was not sufficiently dried, or if the boxes were covered. In this case the larvae leave the food prematurely as 1st or 2nd instar larvae. The parents may may be drown. You may put in a filter paper or transfer with a spoon part of the fluid food to a new vial.
The limiting factor for the number of progeny is usually the amount of food. In single fly crosses, pay attention to the dryness of the food and use rather fresh vials. Supplementing the fresh vials with a drop of liquid yeast accelerates the generation time especially if not overcrowded and significantly increases the no of progeny. Attention: Too much fresh yeast is dangerous, since flies like to stick to the yeast and vials may get slimy if the yeast is not eaten up.
Filter paper is added only after the larvae wander out of the food. The filter paper increase the surface area for pupae and prevent the ecclosing flies from drowning.
Generation time: (duration from egg laying to ecclosion):
Temperature
period
18ºC
 3 weeks
RT
 12-14 days
29ºC
 9 days
Development usually takes longer in crowded vials.

2. crosses:


collecting virgin flies
Since females may store sperms over several weeks, it is absolutely required for controlled crosses to separate newly ecclosed male and female flies before they mate. Although young females mate already within hours after ecclosure, their male siblings do this after a peroid of time that depends on the temperature. You either collect females immediately after ecclosure (visible at the "green" contents in their abdomen - larval gut) or you regularly separate young males and females in intervals according to following table.
Temperature
 Zeit
18ºC
 less than 24 h
22ºC
 ca. 12 h
25ºC
 ca. 8h
29ºC
 ca. 6h

Rule for collecting virgins Remove all flies from the vial. Kill the flies sticking to the food with the head of the brush.
Keep the vials at 18°C over night, at RT during the day
collect virgins in the morning and the late afternoon. You may also collect virgins only once per day. In this case make sure that the temperature is really 18°C and use a 20h period.
Attention: After collecting the virgins, check each vial again for hidden males.
For critical experiments, collect the virgins in several vials and age them to check for larvae. Virgin females lay eggs although in lower numbers.
The ruin of virgin collection is a single male, which can cause a big damage, since they can mate 3 time a day. In contrast females only mate every 2-3 days.

Males
The age of the males does not matter. However they should be in good shape. The number is not so important and can be in surplus. Males are more sensitive to becoming steril, if they are anaethesised for too long with ether or CO2. Similarly, high temperature damage the fertility of males - 29°C is the upper limit.

Crosses
Males and females are put together into a vial, all remaining happens by itself. Anaethesised flies tend to stick to the food/yeast, especially if wet. Simply lay the vials to the side or put them up-side-down into the box until the flies wake up. For setting up new stocks, use small number of parents or even multiple single crosses, to reduce the problem on non-virgins.

3. Eggs and embryos


Flies: The females should be kept in good shape and well fed with fresh yeast of the apple juice plate. The flies start laying eggs after one to two days on yeast. Without yeast the flies will immediately stop laying eggs. As a rule of thumb the yield is 20-100 eggs per female per day, depending on the genotype. Young flies start laying eggs after the second day, highest rate is after 3 to 10 days. Flies prefer egg-laying in the dark and in the afternoon. In principle the light-dark cycle of the incubator could be changed, however it takes some time for the flies adjusting, making it cumbersome.

Apple juice plates: The agar plates should be wet. Dry plates can be improved with a few drops of 5% acidic acid. Yeast is absolutely required. Spread the yeast paste with your finger on the plate, amout depending on the number of flies in the cage. Dry until the surface has lost the glossyness. The eggs should be on the surface of the agar plate. They are too soft, if the flies press their eggs into the agar.

laying periods: Plates should be changed before larvae hatch, which depends on the temperature
18°C ca. 40h    1,5 days
25°C ca. 22h    daily
29°C ca. 18h    twice a day
For synchronised egg collections, the periods can be down to 15-30 min. Shorter periods are not practicable, since females retain the eggs.


Cages: Choose the size according the experiment and amount of eggs needed.
a) big cages (95 mm): if many eggs are needed from a single or few genotypes or for injection experiments with well staged embryos. There should not be more flies in the cage than what is needed to cover the area of the agar plate. To increase the number attach a filter paper to the cage. Make sure it does not touch the agar plate. If the eggs are not collected you may reuse the agar plate, eg for overnight feeding.
Overcrowded cages get wet. The flies will stick to the wall and to the agar plate. The yield of eggs will be lower.

4. Clones

germ-line clones:

Chromosomenes:  (siehe S. Luschnig, Dissertation 2000, Genetics 167 (2004) 325-342
hs-Flp122 (X):  P[ry+, hs-Flp]122
Frt9-2:  P[>w+>, Frt]18E
Frt40A:  P[ry+, hs-neoR, Frt]40A
FrtG13:  P[>w+>, Frt]42B
Frt2A:  P[>w+>, Frt]79DF
Frt82B:  P[ry+, hs-neo, Frt]82B

Stämme:
w f B Frt[18E] hs-Flp122
ovoD Frt[18E]/C(1)/Y
al dp b pr Frt[40A]
Frt[42B] c px sp
y w Flp122 ;  ovoD2L{w+} Frt[40A] / If / CyO, hs-hid{w+}
y w Flp122 ;  Frt[42B] ovoD2R{w+} / If / CyO, hs-hid{w+}
ru h th st Frt[79D]
Frt[82B] cu sr es ca
y w Flp122 ; ovoD3L{w+} Frt[79D] / CxD / TM3, hs-hid{w+}
y w Flp122 ; Frt[82B] ovoD3R{w+} / CxD / TM3, hs-hid{w+}

Cross 20 to 50 virgins (  xxx Frt / CyO, hs-hid)  with a similar number of males (ywFlp122/Y; ovoD / CyO, hid) in a big vial with yeast. Flip into new food vial every two to three days at 25°C. Heat-shock for each 1 h in a 37°C water bath at 24-48 and 48-72 hours. This procedure was optimised to highest proportion of clonal eggs in a non-selective procedure (5% are clones). Later and shorter heat-shocks a also fine, since the clones are selected by ovoD. Using the CyO, hs-hid or TM3, Sb hs-hid balancers selects the flies with clones/kills the balancer flies during the heat-shock.

Selection for the NeoR marker:

Some of the FRT chromosomes (as well as some other transgenes) carry a dominant neoR marker that confers neomycin resistence to the flies. The marker is selected for by simply adding G418 to the fly food. The selection is usually complete, if the culture are not overcrowded. However, the yield of flies is lower than normal.
  • stock solution: 25 mg/ml in water, store at -20¡C. Cell culture quality of G418 is not required.
  • small vials (ca 10 ml): 0.2-0.3 ml
  • large vial (ca 50 ml): 1-1.5 ml
  • procedure: with a needle make a cross-wise slit into the food. Pipette the stock solution into the slit. Wait until the solution is soaked by the food.




Clones in the follicle epithelium of the ovary:
Young (2-3 d) females are incubated for 1 h at 37°C. After 2-4 days at 25°C the clones have a size of 2-4 cells, after 3-5 days 4-8 cells. Persistent clones (clones in the stem cells) are obtained after 7 days.
A good reference for follicle clones is: Margolis and Spradling , Development 121 (1995) 3797-3807).
Frt chromosomes with good markers are available for all chromosome arms, such as nlsGFP, lacZ.

5. Mutagenesis

Ref:  Lewis and Bacher, Methods of feeding ethyl methyl sulfonate (EMS) to Drosophila males. DIS 43 (1968) 193.
Mutagen: 
Ethylmethylsulfonate (Sigma), quite stable, very toxic,
use concentrations of 25-45 mM EMS (should yield in 1-2 lethals per chromosome)
EMS: MW=124 g/mol, d=1,206 g/ml
Detoxification:  Natriumthiosulfat

Young males, aged at 18°C, are starved on water-saturated paper towel (should not be too wet) for 4h. With a syringe mix the viscous EMS with 1% glucose solution (at least 10x through a thin canula, 25 mM: 240 µl in 100 ml). Put a paper towel into an empty big food bottle and  apply the EMS-glucose solution with the syringe drop by drop. Transfer flies into these vials and keep at RT overnight. Transfer the males into a new food bottle for about six hours before they are mated in yeasted big food vials with an equal to half of the number of virgins (fed with yeast on apple juice plates overnight). Some of the males will die by the EMS treatment. Keep the crosses at 25°C and flip over daily. The males are removed after three days.
Incubate everything that has been in contact with EMS in a big beaker of Na-thiosulfat (10% in water, stable for >1 week). EMS has a half-life time of a few hours only in water. After deactivation everything can be normally deposited down the drain or the dustbin.

Efficincy of mutagenesis:
Cross each of about 100 virgin F1 females (X*/FM7) with 2 males (FM7/Y). Check how many of the crosses have no males (X*/Y) among the progeny.
Assuming a Poisson distribution the average number of lethal hits can be calculated from the number of lethal lines.

Protocol:
Day 1:
males
1. Flies are starved on water soaked tissue for 4h (about 100 males per big food bottle)
2. transfer flies to a new bottle with a piece of dry tissue
3. prepare 50 ml 1% glucose
4. transfer approx. 10 ml of glucose to a beaker
5. carefully open the EMS bottle
6. using a 1 ml syringe transfer 0.15 ml EMS into 10 ml of glucose, slowly dispense
7. using the syringe triturate the EMS drops until dissolved in glucose solution (5-6 times)
8. mix with remaining glucose solution
9. transfer about 4 ml of EMS/glucose to the bottles with the flies
10. keep flies in a quiet place at RT overnight
11. females: feed them well on apple juice plates with yeast
Day 2
11. transfer males to a new food bottle for 6 h
12. transfer males to females in a yeasted food bottle

transfer flies to new yeasted food bottle every day
remove males 3 days after EMS application
take care about bottles, transfer larvae to new bottle, if overcrowded


5. Stock collection

Stocks at 18-22°C
The stock collection is maintained at 18-20°C and 50-60% humidity. At a higher humidity the risc of a mite invasion strongly increases. The stocks are kept in three copies that are 0-2 weeks, 2-4 weeks and 4-6 weeks old positions from left to right. 2 x 13 stocks are kept in each box. The flies are transfered into new vials forthnightly. Please stick strickly to the calender! Please use fresh food vials with a big drop of fresh yeast suspension. It takes only a few minutes for the suspension to dry (the surface changes from a glossy to a smooth appearance). Do not use a ventilator. Remove the vials of weak stocks and transfer them to the Kummerbox that is kept at room temperature for recovery. Put an empty vial as a spacer into the box of the stock collection.
Do not forget to transfer the labels! Make sure that they are fully attached to the vial.
When removing flies from the stock collection take the oldest vial and ensure that the other two vials are in good shape. Never remove all three vials and never return the vials to the collection. Stick the label of the removed vial to the latest vial.

Kummerkiste: Weak stocks are kept in the Kummerbox at room temperature. The flies are transfered to fresh vials more regularly, depending on the stocks. Several vials are kept for each stock.
Generel rules:    Be absolutely careful with the stocks. Several of the stocks are unique and can not be obtained anywhere else.
Never return any anethesised flies back to the stock vials!
Never remove all three vials of one stock. If there are no flies in the third vial, wait until the next transfer
Never leave the vials open, e. g. when adding the yeast suspension
Never return the stock vials to a false position
Balancer stocks, wild-type and other useful stocks:
This collection is kept at room temperature in big food vials. The stocks are flipped over every Monday. Supplement the food vials with yeast for the balancer stocks, for the wild-type flies (OrR, w, yw) use plain food vials. Remove the flies from the vials of the previous week.

6.  Common genetic marker

1. Chromosome


Locus
  map-pos.
   Pheuml;notype
y  yellow
 0
 bright/yellow cuticle and bristles
w white
 1.5   
white eyes
pn prune
 ??   
dark red eyes
N   Notch
 3
 wings with notches at the edge, rezessiv letal
cv  crossveinless
 14
 the two cross veins are missing
sn singed
 21
 bristles on the notum are wavy/singed
v  vermillion
 33
 bright red eyes(as cn, st)
f   forked
 57
 kincked and branced bristles
B  Bar
 57
 narrow eyes (B/+) or slits (B/Y or B/B)
    
          

2. Chromosome

Locus
map-pos.
 Phenotype
al
 aristaless
 0
 brush of antenna is short (Arista)
dp
 dumpy
 13
 short wings
b
 black
 48
 dark/black cuticle
pr
 purple
 54
 dark red eyes
cn
 cinnabar
 57
 bright red eyes (as v, st)
sca
 scabrous
 67
 rough eyes
vg
 vestigial
 67
 short wings
c
curved
 75
 wings bended down
px
plexus
 100
 branched wing veins
bw
 brown
 104
 brown eyes
sp
speck
 107
 dark spot at wing basis
S
 Star
 1
 rough eyes
Cy
Curly
 6
 wings curled
Sp
 Sternopleural
 22
 more than 3 bristles at sternoplurum (ts!
Sco
 Scutoid
 51
 reduced no of brisles on scutellum
Tft
 Tufted
 53
 more bristles on notum and scutelum
Bl
 Bristle
 55
 short bristles (ts!)
L
 Lobed
 72
 small eyes
If
 irregular facets
 107
 eyes half size


3. Chromosome

Locus
map-pos.
 Phänotyp
ru
 roughoid
 0
 rough eyes
h
hairy
 27
 hairy wing veins
st
 scarlet
 44
 bright red eyes (as v, cn)
ri
 radius incompletus
 47
 outer wing vein missing
p
 pink
 48
 dark eyes
cu
 curled
 50
 like Cy
sr
 stripe
 62
 sark stripe on notum
e
 ebony
 71
 dark cuticle
ca
 claret
 101
 dark eyes
Me
 Moire
 19
 Augen schimmernd
Ly
 Lyra
 41
 defect edges of wing
D
 Dichaete
 41
 wings go away from fly
Hu
 AntHu
 47
 bristles on propleurum
Sb
 Stubble
 58
 Borsten kurz, stumpf
Ubx
 Ultrabithorax
 59
 Haltere vergrößert, mit Borsten
Pr
 Prickly
 90
 Makrochaeta fehlen
Tb
 Tubby
 91
 Larven, Puppen (junge Fliegen) Pkurz und dick
Ser
 Serrate
 93
 Flügelspitzen eingeschnitt
Dr
 Drop
 99,2
 kleine bis keine Augen



7.  Balancer

1. Chromosom:

M5 Muller 5 sc wa B
 für letale auf dem X, lebensfähig und fertil
FM3 First multiple 3 y  sc dm B
 für maternal-effects auf dem X, letal in Männchen
FM6


FM7 First multiple 7 wa sn  v  g B für letale auf dem X, lebensfähig in Männchen, steril in FM7/FM7 Weibchen, schwach gelbe Augen
FM7c
y wa B
 

2. Chromosom: alle homozygot letal

CyO Curly of Oster Cy, dp1vI, pr, cn special CyOs with b, bw, DTS, heatshock-hid{w+}, hb-lacZ{ry+}, GFP
SM1 Second multiple 5 Cy, al2, cn2, sp2 balances better than CyO, in particular at the tips of 2L (al) and 2R (Kr)
SM5
Cy, al2, ltv, cn2, sp2 does not go well at high temperature
SM6B
Cy, Roi
In(2L2R)
Gla Bc


3. Chromosome

TM1
Me, ri sbd2 also with cu or p, goes well, Me difficult to see
TM3
Ser, ri, pp, sep, bx34e, es also with Sb and Ser, and Sb, Ser, st, ri, p, e (besser, da Ser manchmal schwierig)
TM3, hb

trägt ein hb-lacZ(ry+) reporter gene
TM3, hid

trägt ein HS-hid(w+) transgene, DTS (1h 37°C), geht auch bei 29°C
TM6B

Third multiple 6B, Hu, e, also with D or Tb; Hu is difficult to see, good balancer
TM6C

Third multiple 6C, many variant also with e, Sb Tb, good balancer
TM2
Ubx, es,p not suited for tip of 3R
 

4. GFP/lacZ Balancer


FM7 ftz-lacZ(w+) erst in stage 7 sichtbar
FM7c actin-GFP starke maternale Komponente, gut für Larven
CyO hb-lacZ(ry+) ab Beginn Zyklus14 sichtbar
CyO actin-GFP(w+)
strong maternal contribution, suited for larvae
CyO Kr-GFP(w+) sure at stage 8, with antibody staining
TM3 Sb hb-lacZ(ry+) visible from mid cellularisation
TM3 Ser actin-GFP(w+) strong maternal expression, suited for selecting larvae
TM6 Ubx-lacZ
TM6 nulloHA weak staining

8.  Microinjektion von Embryonen

  • collect staged embryos
  • prepare cover slides with glue with a pasteur pipette
  • collect embryos in a net
  • dechorionate with 50% bleach for 90s, wash with tap water
  • cut a  slice of apple juice agar place on  a glass slide, agar should be dry
  • transfer embryos with a fine brush
  • align embryos on the agar slice with a preparation needle, carry them do not roll!
  • align at the edge of the agar slice in a mirrow image orientation
  • gently place the cover slide with the glue on top of the embryos, press gently with the preparation needle
  • take of the coverslide (with the attached embryos)
  • try in a dessication chamber for 5 to 10 min
  • cover embryos with 10S voltalef oil
  • inject with a 20x (or 10x) objective

Setting of the Eppendorf pressure maschine

(depends on your experimental conditions) an expample:
Pi 1000
ti 0.3-0.5
Pc ca 50

glass capillaries:

Depending on how they are loaded you use capillaries with or without internal filament. They capillaries are purchased at World Precision Instruments or preferably at Biomedical instruments/Gündel. The needles are prepared with a needle puller with settings depending on your taste. Please be careful with the heating filament. Before use the tip of the needle is broken with a fine pair of tweezers at the region of the tip where you can see the colour spectrum.
Borosilikat, outer diameter 1 mm, inner diameter 0,78 mm, length 100 mm

9. Mikrosporida:


Microsporidia are unicellular parasites that infect mostly gut cells of insects. Transmission of the infection is by spores that are secreted through the gut. In case of Drosophila transmission from stock to stock is most likely by mites. Infection by microsporidia strongly decrease the viability and fertility. Flies die after only few days. Dead flies are found laying on the food in flipped-over vials within a week. Adult flies have big abdomens. In strongly infected stocks the are many black third instar larvae and black pupae visible. The infection is transmitted for example by larvae eating dead adult flies. Transmission from stock to stock is mostly by mites.

Treatment: It is essential to prevent spreading of the infection within the stock collection by strict mite control. Chemical treatment by fumagillin added to the food was not successful. Fumagillin inhibits the new formation of spores. Treatment of embryos with bleach proofed to be most efficient.

Add bleach to a overnight collection of eggs on a small apple juice plate. Incubate for several minutes. Collect embryos in a net. Wash with water. Add water to the plate, mobilise remaining embryos stuck in the agar with a brush. Collect in a net. Make sure that no larvae are transferred which will already have eaten some spores. Incubate the embryos in the net for a second-time in bleach. Make sure all embryos have been treated with bleach and that nothing is transfered that was not incubated with bleach. Wash with water, dry on a paper towel, transfer embryos with a brush to a new agar plate. Cover the embryos with little voltalef oil. Incubate at 25°C. Collect the larvae as soon as they hatch with a needle and transfer them to a fresh food vial. Collect about 50-100 larvae. Incubate at 25°C. After ecclosion check the genotype of the flies.


10. Wings, cuticles, guts, imaginal discs


Wings

With a razor blade cut both wings off from an anaethesised fly hold by tweezers with the other hand. Before mounting in a drop of Hoyers:lactic acid the wings was briefly washed in Ethanol for a minute and dried on a piece of paper. Before adding the coverslide try to get ride of the air bubbles. Incubate at 65°C with a weight on top to flatten the wing sample. The full wing is observed at the microscope at 4x with the condensor lense on the side. Sections are observed at 40x bright-field or DIC.

Cuticule preparations

Collect over-aged embryos and transfer into a net where they are washed, dechorionated and washed with water. Transfer with a fine brush the embryos to a drop of Hoyer:lactic acid on a clean slide. Push the embryos into the Hoyers and cover with a clean coverslide. Incubate for several hours/overnight at 65°C. Add a piece of metal to press the specimen, if prefered. Observe with dark field optic or phase contrast.
For nice preparations the vitelline membrane may be removed. Embryos are fixed as usual in 4% FA/PBS/heptan. After poping with methanol and rehydration in PBS, the fixed embryos are mounted in Hoyers/lactic acid. Alternatively, the coverslide is shifted back and forth after mounting. For special preparations, the embryos may be released from the viteline membrane manually, similar as it is done with injected embryos.

Hoyers: 
Dissolve 30 g Gummi arabicum (Merck) with 50 ml deionised water in a beaker by stirring overnight. Make sure everything has dissolved. While stirring add stepwise and slowly 200 g Chloralhydrat to prevent clumping. After further addition of 20 g Glycerol (98%), centrifuge for >1 h at 12000xg). For nice dark field preparations is is essential to have an extremly pure Hoyers solution.
Before mounting mix equal volumes of Hoyers and lactic acid. Mix and wait until the solution is uniform. You may store this mixture for several weeks. Clean the slide and coverslide - every bit of dust will be seen in darkfield optics. Clear the preparation at 65°C for several hours. Reference: H. Hoyer. Beiträge zur histologischen Technik. Biol Cent. 2 (1882) 17-24.

Imaginalscheiben aus Puppen:

Puppen im richtigen Stadium werden vor und während der Präparation fixiert. Nach der Freilegung der Scheiben müssen diese noch aus dem Kutiklulasäckchen befreit werden. Für Immunofärbungen werden die Scheiben mit 0,2% Triton permeabilisiert.
weiße Puppen in ein neuen Röhrchen überführen, altern lassen. Y. Ahmed, Dartmouth, Hanover, USA)
  • mit der Mikroschere die vordere und hintere Spitze der Puppe abschneiden
  • in einem Uhrglas für 20 min mit 4% Formaldehyd/PBS mit ein wenig Heptan fixieren. Die Fixierlösung kann auch mit einer 28G Kanüle injiziert werden (10µl Lebensmittelfarbe in 1 ml Fix).
  • die fixierte Puppe wird in der Mitte durchgeschnitten, um das Abdomen zu entfernen. (Schere und Pinzette)
  • die Puppenhülle wird mit der Schere aufgeschnitten, so dass die vordere Puppe frei wird (Schere und Pinzette)
  • die Flügelscheiben werden freipräpariert und in ein anderes Uhrglas mit PBT überführt und dort gesammelt (Schere und Pinzette)
  • die Scheiben werden aus dem Kutikulasäckchen befreit (Pinzette und Wolframnadel) und im Eppi mit PBT gesammelt
  • zur Blockierlösung werden 0,2% triton-100 zugegeben, um das Gewebe zu permeabilisieren. Für f-actin und DNA Färbung ist dies nicht notwendig.




11. Misc


Tungsten needle:

The needles are sharpened electrolytically. Put a tungsten wire connected with a cathode into a 10% KOH solution. The anode is a simple wire (e. g. a office clamp) Apply for seconds to a minute a voltage of 12 V. Check the sharpness of the tungsten wire with the stereomicroscope.
Attention: 10% KOH dissolve the skin!
Source: Goodfellow GmbH, Bad Nauheim, Wolfram-Draht, 0.2 mm diameter.
Reference: J Brady, A simple technique for making very fine, durable dissecting needles by sharpening tungsten wire electrolytically. Bull World Health Organ 32 (1965) 143-144

Mounting media


Mowiol:

glycerol2,4 g
Mowiol 4-88
 6 g
dH2O
 6 ml
0,2 M Tris/HCl pH8,5
 12 ml

Fill glycerol into a 50 ml disposible conical centrifuge tube. Add Mowiol and stir thoroughly. Add water and incubate for 2 h at RT. Add Tris solution and inucbate at approx. 53°C until the Mowiol has dissolved completely (take years!), mix occacionally. Centrifuge at 4000-5000 rpm for 20 min and aliquot in eppendorf vials. Store at -20°C, stable for >1a. Before use add some crystalls of DABCO (2,5%). Use within days.
Advantage of mowiol: The medium retracts, so that the specimens gets thinner.
Mowiol: Poly(vinyl alcohol- vinyl acetate), Polyscience Cat 17951, 500g
DABCO:  (1, 4 - diazobicyclo-[2,2,2]- octane
 


Aqua-Polymount, DS#432

store at 4°C, if too thick, you may dilute with water
ventor:
Polysciences Europe GmbH,
Kundennummer:  30920
Art. Nr.: 18606-20
Preis:  56,90/20 ml Flasche

Glue for embryos:

Cut about 1 to 2 m of tape (TesaPack, some of the other tapes do not work equally well) in small pieces and shake or rotate with about 20 ml n-heptan in a 50 ml falcon tube for overnight. Remove the heptan suspension and add new heptan if necessary. To remove insoluable material spin the heptan suspension in the ultacentrifuge for 30 min. Save the clear supernatant, distribute in aliquots, best in scintillation vials that are air-tight. Discard the insoluable material and carefully clean the centrifugation tubes. If the glue is not completely clear, repeat spinning.

Einbettung und Schnitte von gefärbten Embryonen

This protocol works for embryos stained with a variety of techniques, like DAB, DAB with heavy metal enhancement, phosphatase substrates. it also works for in situ hybridized embryos, it they are refixed before tranfered to aceton. It does not work very well for flourescently labelled embryos, because the araldite is slightly flourescent.
  • dehydrate embryos through a methanol or ethanol series in eppendorf vials
  • incubate 2x10 min in 100% ethanol
  • exchange with dry acetone
  • exchange with aceton-araldite mix (1:1)
  • incubate for several hours or overnight
  • transfer embryos with a cut yellow tip to a slide or a small snap cap
  • let the aceton evaporate for some time. The embryos can be kept in araldite at 4°C for a few days, at -20°C for a very long time (>1 a).
  • transfer embryos with a tungsten needle to a slide for mounting or to the rubber template for sectioning blocks. Arrange embryos
  • Incubate at 60°C for a short while. Rearrange embryos in the blocks. Arrange them to the narrow end of the mold as much as possible. The closer you get, the less araldite you will have to trim before sectioning.
  • polymerise araldite at 60°C for one day
  • insert polymerised block into the microtome holder. Wtih a razor blade trim block to a small pyramid around the embryos o the shape shown below.

Araldite: (Durcupan ACM from Fluka/Sigma)
The quality of sections depends to a large extent on the quality of the araldite, so it is imperative to prepare the araldite very carefully (precise amounts, good mixing, patience). Careful, Araldite is not very healthy, wear gloves.
in a glass beaker stir 54,33g of reagent A with 47,41 g of reagent B until completely mixed. Slowly stir in 3.5 ml of reagent D. slowly add 2 ml of reagent C
Sigma:
A/M
Epoxyharz100 ml44611
BHärter100 ml44612
CBeschleuniger100 ml44613
DWeichmacher100 ml44614

Aceton:
  • activate molecular sieve (3A) by heating overnight
  • add beads to aceton bottle
  • store at RT


food vials and plugs

The plasic vials and corresponding plugs are purched at Greiner and Klühspies, respectively.

type size
(mm) 		number price
(Euro) 		price/piece
Greiner
vial 26 1000x 44,40 4,44c
plugs 26 1000x 43,97 4,4c
tin 50 1000x 203 20,3c
plug 50 1000x 90,50 9c
Klühspies
vial 28 1000x 58 5,8c
plug 26 4500x 405 9c
plug 35 - - -
tin 49 1000x 191 19,1c
plug 50 1000x 200 20c