Resin Embedding, Sectioning, and Immunocytochemical Analyses of Plant Cell Walls in Hard Tissues - Plant Cell Morphogenesis: Methods and Protocols - page 47

3. Incubate with PBS for 5 min.
4. Incubate with primary MAB diluted in PBS/MP for at least
1 h at RT or overnight at 4 °C. A 5-10-fold dilution of a
hybridoma cell culture supernatant is a good starting point for
the primary MAB; however, a range of dilutions should be
assessed ( see Note 8 ). For overnight incubations it is useful to
incubate slides in a sealed container on wetted fi lter paper to
prevent drying out.
5. Wash with three changes of PBS with at least 5 min for each
change.
6. Incubate with a secondary antibody diluted in the region of
100-fold in PBS/MP for at least 1 h at RT. For example, anti-
rat IgG (whole molecule) linked to FITC is widely used for rat
MABs. Alternatively anti-rat IgG linked to AlexaFluor 488
may be used, which is brighter and more photostable.
7. Wash with three changes of PBS with at least 5 min for each
change.
8. Incubate with a 10-fold dilution of the Calcofl uor White stock
solution for 5 min ( see Note 9 ).
9. Wash with three changes of PBS, each 5 min.
10. Mount samples using a small drop of anti-fade reagent (for
Multitest slides 2 l is a suitable volume), cover with a cover

slip, and examine. To prevent slippage and slides drying out,
the edges of the cover slip can be sealed.
11. Examine with a microscope fi tted with epifl uorescence optics
and fi lters (e.g., UV, FITC, and TRITC). Sample autofl uorescence
can be assessed by examining the no-primary-antibody-control
in the FITC and TRITC channels ( see Note 10 ).

μ

1. Isolate individual wells on slides and block nonspecifi c binding
sites ( see Subheading 3.4 , steps 1 and 2 ).
2. Incubate with the CBM diluted in PBS/MP for at least 1 h at
RT. The most effective working concentration should be
determined by trial studies for each CBM, but most CBMs can
be used effectively in the range of 5-20

3.5 Immunolabelling
of Plant Cell Walls
Using Recombinant
CBMs

g/ml.
3. Ensure that there is a no-CBM-control to assess cell wall
autofl uorescence in the section.
4. Wash with three changes of PBS, each 5 min.
5. In the case of a CBM fused with a fl uorescent protein, proceed
directly to step 7 . In the case of a CBM with a His tag, incu-
bate with anti-His coupled with FITC at 100-fold dilution in
MP/PBS for at least 1 h. Alternatively anti-His linked to
AlexaFluor 488 may be used.
6. Wash with three changes of PBS, each 5 min.

μ