Developing sporophyte
This is a not so great axial cross section of a developing sporophyte while still part of the gametophyte. The sporophyte growth would have been to the right. This material proved very difficult to section and I would have preferred a more central section.
The following protocol is largely based on Chamberlain, "Methods in Plant Histology, 5th ed., 1905. Fixed 24 hours in aqueous chromic acid-acetic acid (3.7 g potassium dichromate/l + 10 ml glacial acetic acid/l). (The substation of potassium dichromate for chromic acid based on equivalent chromate conc. is reported to work as long as the pH is less than 3.4). Washed 24 hours in flowing water. Dehydrated in 10, 35, 50, 75, 85, 95, 99 % IPA in water, min 6 hours each. Cleared in IPA:Xylene at 3:1, 2:1, 1:3,1 hour each and pure xylene for 10 min. Infiltrated in xylene saturated with Paraplast for 5 days, followed by 3 changes of melted Praplast for 2 hours each. Embedded in Paraplast. Sectioned on a Spencer 820 microtome at 7 micron. Cleared in Xylene 2X, 10 min each. Rehydrated 99, 95, 70 % IPA, followed by water for 5-10 min each. Stained in Gills Haematoxylin, 5 sec, water washed 5 min. Stained 1% Eosin, 1 min. Dehydrated 2X 99% IPA, 2 MIN. Cleared 2X xyene 2.5 min each. Mounted with DEPEX.
Photographed on a Nikon MS microscope at 200X original magnification using a Sony NEX 5N with a Leica MIKAS 1/3X adapter.
Developing sporophyte
This is a not so great axial cross section of a developing sporophyte while still part of the gametophyte. The sporophyte growth would have been to the right. This material proved very difficult to section and I would have preferred a more central section.
The following protocol is largely based on Chamberlain, "Methods in Plant Histology, 5th ed., 1905. Fixed 24 hours in aqueous chromic acid-acetic acid (3.7 g potassium dichromate/l + 10 ml glacial acetic acid/l). (The substation of potassium dichromate for chromic acid based on equivalent chromate conc. is reported to work as long as the pH is less than 3.4). Washed 24 hours in flowing water. Dehydrated in 10, 35, 50, 75, 85, 95, 99 % IPA in water, min 6 hours each. Cleared in IPA:Xylene at 3:1, 2:1, 1:3,1 hour each and pure xylene for 10 min. Infiltrated in xylene saturated with Paraplast for 5 days, followed by 3 changes of melted Praplast for 2 hours each. Embedded in Paraplast. Sectioned on a Spencer 820 microtome at 7 micron. Cleared in Xylene 2X, 10 min each. Rehydrated 99, 95, 70 % IPA, followed by water for 5-10 min each. Stained in Gills Haematoxylin, 5 sec, water washed 5 min. Stained 1% Eosin, 1 min. Dehydrated 2X 99% IPA, 2 MIN. Cleared 2X xyene 2.5 min each. Mounted with DEPEX.
Photographed on a Nikon MS microscope at 200X original magnification using a Sony NEX 5N with a Leica MIKAS 1/3X adapter.