Selected Simple Methods of Plant Cell Wall Histochemistry and Staining for Light Microscopy - Plant Cell Morphogenesis: Methods and Protocols - page 30

2.2 PARS Reaction

1. Periodic acid solution: 1 % (w/v) H 5 IO 6 . Signifi cantly lower
concentration (0.2 %) proved to be also effi cient.
2. Schiff 's reagent according to de Tomasi: Dissolve 1 g of basic
fuchsine in 200 ml of boiling distilled water. Stir the solution
for 5 min and let it cool down to 50 °C, fi ltrate with paper.
Add 20 ml of 1 M HCl. Cool down to 25 °C. Add and dis-
solve 1 g of Na 2 S 2 O 5 (potassium metabisulfi te). Leave in dark
for 14-24 h to gain a pale yellowish-orange clear solution.
Add enough active coal (approx. 2 g) and shake for few min-
utes, fi lter on paper to gain clear colorless solution. Store in a
dark tightly stoppered bottle at 4 °C. The solution deterio-
rates with time. Discard when it turns colored.
3. SO 2 water: Mix 5 ml of 1 M HCl with 5 ml of 10 % K 2 S 2 O 5
and 100 ml H 2 O before use. Solution remains effi cient for a
few days in a closed bottle.
4. Reducing solution: Dissolve 1 g of KI and 1 g of Na 2 S 2 O 3 .5H 2 O
in 50 ml of H 2 O, add 0.5 ml of 2 M HCl. Prepare fresh before use.

1. Primary antibody solution: Dilute monoclonal antibody
towards (1-3)-

2.3 Callose
Immunodetection -glucan (Biosupplies Australia PTY Ltd) 1:100

in 1× PBS with addition of 10

β

μ l of BSA stock per 1 ml of fi nal

solution.
2. Secondary antibody solution: Select anti-mouse or anti-rabbit
IgG antibody of your choice (we use Invitrogen anti-mouse IgG
Alexa Fluor 488; 1:1,000) and dilute accordingly in 1× PBS
with addition of 10

3. 10× PBS (phosphate-buffered saline) stock solution: Weight
80.1 g NaCl, 2 g KCl, 14.7 g Na 2 HPO 4 . 2H 2 O, and 2.38 g
KH 2 PO 4 to prepare 1 l of solution. 10× PBS stock has pH 6.8,
after dilution to 1× PBS should be pH 7.3. It is recommended
to check with pH meter before use.
4. 10 % BSA (bovine serum albumin) stock solution: Dissolve
1 g of powdered BSA (Fraction V) in 10 ml of distilled H 2 O.
Store in 1 ml aliquots at −20 °C.
5. Casein 3× stock solution: Add 3.33 % (w/v) of casein into
distilled water and titrate to pH 10 with minimal amount of
2 M KOH. Let casein to dissolve at 40 °C with constant stir-
ring (approx. 2 h). When it is completely dissolved, titrate to
pH 7 with minimum of 2 M HCl. Add 10× PBS stock (11 %
of volume of solution prepared aforetime) to gain 3 % casein
solution in PBS. Aliquots can be stored at −20 °C. Before
usage dilute (1:2) with PBS.
6. Buffered glycerol with n-propyl gallate: Add 3 % (w/v) of
n-propyl gallate (antifade reagent) into glycerol and stir over-
night at room temperature (it is not readily soluble in aqueous
solutions). Mix 8:2 with TRIS buffer (0.1 M, pH 9.0).

μ l of BSA stock per 1 ml of fi nal solution.