Washing and Mounting

The second method is also very convenient and rapid once the labeled primary antibodies have been produced. A cocktail of the two (or more) fluorophore-tagged primary antisera is applied to sections or whole tissues, and after washes and mounting they are ready for microscopical analysis... 

Fig. 1. Set up for sample staining and washing. Materials for cell staining, washing, and mounting coverslips are shown.

Rinse tissue three times in Sodium Phosphate/TX (10 minutes each wash) and mount in antibleach medium (step 7a). 

Fig. 2 Washing and mounting of brains, (a) Immunostained brains are washed in mesh-bottomed inserts of 12-well plates on a rocking platform. The /rrsef shows top and bottom views of the insert, (b) An immunostained brain is mounted in LMP agarose on a glass coverslip for confocal imaging, (c) The brain embedded in LMP agarose. Scale bar, 2 mm. The inset Is a closeup dorsal view of the brain. Anterior is to the top...

Another method is the denaturation of a tissue section with denaturant on the membrane. In this method, the frozen section is thawed and mounted on the membrane. The transferred membrane is washed with 70% ethanol to remove salt and lipid in the tissue and to fix the protein on the membrane. After that, denaturation is processed with the denaturant. Another method is... 

Gun Design, Chapt 2—Gun Construction, Chapt 3—Recoil, Chapt 4-Carriages and Mounts and Chapt 5—Breech Mechanisms) 56)Anon, "Artillery Ammunition, Dept of the Army Tech Manual, TM 9—1901, Washington,DC(1950) 57)ORDIM,Office of Chief of Ordnance Washington 25,DC,"Com-plete Round Charts, No 5981(1950-1) 58)Anon, "Ordnance Proof Manual, Nos 1—1 to 70-10, years of issue 1937-1951, Office of Chief of Ordnance, Washing ton,DC 59) R.Held, "The Age of Firearms, A Pictorial History, Harper Bros, NY(1951) 60)C.R. Jacobs, "Official Gun Book,1 CrownPublg Co,NY(1951) 6l)Anon, "Ordnance Inspection Handbook on Ammunition Material, ... 

DAB- Cover the section with this substrate, and incubate the slide for 5 min The product is brown, and is stable m alcohols and in xylene. Counterstain with Mayer s hemalum for 5 mm Wash in tap water. Dip the slide in saturated lithium carbonate for a few seconds this makes the nuclear stain blue. Wash m tap water Dehydrate through ethanol and xylene (or Histoclear), and mount in a permanent mountant, e.g, DPX... 

Wash well in tap water, dehydrate in IMS, clear in xylene, and mount in DPX... 

All incubations in antibody/avidin/enzyme conjugates described below are carried out at room temperature for 30 min unless otherwise stated. The substrate reactions are carried out at room temperature, and signal development monitored by light microscopy. The substrate incubation times are, therefore, determined empirically for each experiment. All slides are finally washed in distilled water, air-dried at 42°C, and mounted in glycerol jelly. 

After excess water is removed, the sections are covered with 200-300 pi of silver enhancement mixture for 8-11 min at 20°C. The silver enhancement mixture is prepared immediately before use by mixing equal volumes of the enhancer and initiator solutions of the Janssen Intense LM kit. The sections are washed three times for 5 min in distilled water, dehydrated, and mounted. 

Wash twice in water, dry and mount in buffered glycerol. 

Step 3. Transfer slurry to tared paper filter in filtration apparatus. Wash out each centrifuge tube with 3 portions of 5 mL of water and pour through filter. Wash and dry with 3 portions of 5 mL ethanol. Place each filter on tared planchet and heat in oven at 100 C for 2 hours. Cool. Weigh. Calculate Pdl2 carrier weight and yield and record in Data Table 10.2 Mount filters for counting. 

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