EM Methods

Contents:

 

Processing Tissue for Spurs Resin (Morphology)

  1. Fix tissue in 4% PFA, 4% sucrose and 0.1% glutaraldehyde O/N
  2. Rinse tissue in PBS
  3. Post fix tissue in 1% OsO4 1 hr
  4. Wash in MQ X3
  5. Dehydrate in graded acetone (30%-absolute), 15 min each
  6. Incubate in absolute acetone 3x 15 min
  7. Incubate in acetone:Spurs resin 1:1 1hr
  8. Incubate in 100% Spurs 1hr X2
  9. Incubate in 100% Spurs O/N
  10. Embed and polymerise O/N at 60˚C

 

Immunolabeling Resin Sections

  1. Incubate sections in 50mM glycine 30min
  2. Pre-incubate in 2.5% blocking serum in incubation buffer 10min
  3. Incubate in primary Ab diluted in incubation buffer
  4. Wash in immuno incubation buffer 6 X 5min
  5. Incubate in biotinylated secondary antibody
  6. Wash in immuno incubation buffer 6 X 5min
  7. Incubate in streptavidin gold
  8. Wash in immuno incubation buffer 3 X 5min
  9. Wash in phosphate buffer 3 X 5min
  10. Fix 5 min
  11. Wash in phosphate buffer 2 X 5min
  12. Wash in MQ 3 X 5min
  13. Stain in uranyl acetate and lead citrate 10min each

NB: Block in serum that secondary Ab is raised in

Controls: normal serum of same isotype and protein concentration as primary Ab

Immunoincubation buffer control

All antibody solutions must be centrifuged before use

All antibody dilutions made in immunoincubation buffer

 

Spurs Embedding Media

 

Low Viscosity

Firm

 

~50mL

~50mL

Vinylcyclohexene (VCD)

10g

10g

Diglycidyleter of polypropylene glycol (DER)

4g

6g

Nonenyl succinic anhydride (NSA)

0.26g

26g

Dimethylaminoethanol (DMAE)

0.4g

0.36g

 

Processing Tissue for Cryosectioning

  1. Fix tissue in 4% PFA, 4% sucrose and 0.1% glutaraldehyde O/N
  2. Rinse tissue in PBS
  3. Incubate in 1.7M sucrose 15% PVP:MQ, 1:1, 1X 2hr
  4. Incubate in 1.7M sucrose 15% PVP O/N
  5. Trim blocks and plunge freeze in liquid Nitrogen
  6. Store blocks in dewer

Immunolabelling Cryosections

  1. Incubate sections in 50mM glycine 30min
  2. Pre-incubate in 2.5%b blocking serum 10min
  3. Incubate in primary Ab
  4. Wash in immuno incubation buffer 6 X 5min
  5. Incubate in streptavidin gold
  6. Wash in immuno incubation buffer 3 X 5min
  7. Wash in phosphate buffer 3 X 5min
  8. Fix 5 min
  9. Wash in phosphate buffer 2 X 5min
  10. Wash in MQ 3 X 5min
  11. Embed in 1% methylcellulose: uranyl acetate, 9:1 10min

 

NB: Block in serum that secondary Ab is raised in

Controls: normal serum of same isotype and protein concentration as primary Ab

Immunoincubation buffer control

All antibody solutions must be centrifuged before use

All antibody dilutions made in Immunoincubation buffer

 

Processing for L R Gold (Immunolabelling)

  1. Fix tissue in 4% PFA, 4% sucrose and 0.1% glutaraldehyde O/N
  2. Rinse tissue in PBS
  3. Dehydrate in graded alcohol 15min each
  4. Incubate in absolute alcohol 3 X 15 min
  5. Infiltrate in abs. alcohol: L R Gold 1:1, 1 hr
  6. Infiltrate in 100% L R Gold 1 hr X2
  7. Infiltrate in 100% L R Gold O/N
  8. Polymerise under UV light O/N

Buffers

0.1M Phosphate buffer (Sorenson's sodium phosphate buffer)

  • A. 0.2M Sodium phosphate monobasic NaH 2PO 4.2H 2O 15.6g dissolved in 500ml MQ
  • B. 0.2M Sodium phosphate dibasic Na 2HPO 4 (anhydrous) 14.2g dissolved in 500ml MQ

pH dibasic with monobasic. Dilute to 0.1M for use. Add azide to 0.02%

Sodium Cacodylate Buffer

  • 0.2M Sodium cacodylate Na(CH 3)2AsO2.3H2O - 21.4g dissolved in 500 ml MQ water
  • 0.2M HCl - Dilute 10ml conc HCl (36-38%) in 603ml MQ - Adjust pH of 0.1M cacodylate buffer with 0.2 M HCl to pH 7.4

Reynolds Lead Citrate Stain

Lead solution (0.19M) Pb(NO 3)2 MW= 331.2

  • Dissolve 3.125g lead citrate in 50 ml boiled (CO 2-free) and filtered MQ

Citrate solution (0.28M) NaC 6H 5O &.2H 2O MW= 294.1

  • Dissolve 4.15g tribasic sodium citrate in 50ml boiled (C02-free) and filtered MQ
  • Add 1 drop of lead solution

Sodium Hydroxide (1M) NaOH MW=40

  • Dissolve 2g sodiumm hydroxide pellets in 50ml boiled (C02-free) and filtered MQ
  • Working solution
    • add 210 ml lead solution to 210 ml citrate solution
    • mix (white precipitate will form)
    • add 80 ml NaOH
    • mix
  • stain for approx 10min in the presence of solid NaOH

Incubation buffer for immunolabelling

Makes 200ml, make into 10ml aliquot's and freeze.

 

 

Final Concentration

NaPO4

20mL 0.1M Stock

10mM

IGSS Gelatine

200 ml

0.1%

Ovalbumin

1g

0.5%

NaCl

1.752g

150mM

NaN3

0.26g

20mM

MQ

180mL

 

For NaPO4 use Sorenson's phosphate buffer recipe, pH 7.4

4% Paraformaldehyde

  • 4g PFA to 95 ml PBS
  • Heat to 56 C for 2 Hr while stirring add 10mNaOH dropwise until solution is clear
  • adjust pH to 7.3
  • add PBS to make up to 100ml

 

Paraformaldehyde/ Gluteraldehyde 0.1%

  • As above but add 5ml Gluteraldehyde before adjusting pH