Recombinant sugar binding proteins and their functional analysis: Galectin-1

 Day 1

Isolate subventricular zone tissue from E14.5 embryos and place in ice-cold culture media until all the embryos required are dissected.

 Add 3 ml of Accumax (supplemented with DNAse at 10μg/ml), incubate in a 37^oC water bath for 10 min, and then dissociate cell aggregates by pipetting up and down with a 5 ml pipette for about 10 times.

 Wash the tissue by adding 10 ml of culture media and centrifuge at 800 rpm (140 g) for 5 minutes.

 Re-suspend the pellet in culture media and perform cell count and viability test (e.g. trypan blue exclusion assay). Viability is usually > 70% (if lower, abort the experiment and repeat from step 1).

 Dilute viable cells in culture media to a density of 0.5 - 1x10⁶cells/ml and incubate at 37^oC, 5% CO₂ (the cells should be mostly discrete single cells)

 Day 4

Add the same volume of culture media as used for plating so that the original volume is doubled.

 Day7

Passage the neurospheres (Figure 1) using Accumax as day 1. At this time point, EGF and FGF-2 responsive NSCs are highly condensed among the cultured cells. Add Galectin-1 to the media immediately after plating the cells at desired concentration. 2ME (10mM) should also be added to maintain Galectin-1's carbohydrate binding activity.

 Day 1

Brains are perfusion-fixed with 4% paraformaldehyde and post-fixed in the same fixative overnight.

 Day 2

Brains are cut into 50 mm sections using a vibratome. Brain Matrix (see above) is useful to trim the brain to mount on the vibratome. Place the sections into a 24 well plate filled with 60%Glycerol/ PBS/ 0.1% NaN₃ solution and keep at 4^oC overnight. On the next day, transfer the plate to a -20^oC freezer until required. The sections can be stored at 20^oC for over 5 years.

 Rinse the sections three times in PBS/ 3%H₂O₂ and then place into citric acid buffer (described above). Heat the sections to 95^oC for 1-4 minutes using a microwave.

 Rinse the sections three times in PBS. Incubate with anti-Galectin-1 primary antibody (see above) in TNB buffer overnight at 4^oC. For double or triple immunolabeling, other primary antibodies can also be applied at this stage.

 Day 3

Wash the sections three times in PBS. Incubate with HRP-conjugated anti-rabbit IgG antibody (see above) and a nuclear dye (e.g., Hoechst 333258) in TNB buffer (see above) for 60 min at room temperature.

 Rinse the sections three times in PBS and perform TSA reaction (see above, also refer to the kit’s instructions) for 10 min.

 Mount sections onto a glass slide using Permafluor (see above). The sections are now ready for examination and analysis using fluorescence microscopy (Figure 2).

 Anesthetize the mouse with a mixture of gas (N₂ 3.5 l/min, O₂ 1.5 l/min, Isoflurane 2 l/min).

 Implant a 28-gauge guide cannula into the lateral ventricle using the following coordinates: 0.2 mm anterior from the bregma, 0.8 mm lateral from the midline, and 2.0 mm deep from the brain surface.

 Load the micropump injector with 56 μl of solution containing CS-Galectin-1 (10 mg/ml) in PBS. Gradually infuse the solution into the lateral ventricle of the mouse brain for 7 days by osmotic pressure.

 To label adult NSCs, administer BrdU (1 mg/ml) to the animal’s drinking water during the period of infusion (7 days).

 The mouse is sacrificed 10 days after the last day of the infusion and the brain is processed for immunohistochemistry and counting (Figure 3).