15.5. 1976

Specialia

B) The recording system. As shown above, recording of pO 2 and APs m u s t be done at the same electrode tip. B o t h APs and pO 2 are low-level m e a s u r e m e n t s which need high gain amplification and a good signal-to-noise ratio. If b o t h m e a s u r e m e n t s are done in one circuit, the i n p u t amplifier should be a perfect current amplifier w i t h v e r y low serial resistance and, at the same time, a good AC-amplifier w i t h low noise amplification (Figure 1). The whole electronic s y s t e m consists of 2 parts: one containing AC, DC amplifiers, potentiometers, meters, power supply and insulation amplifiers, and one other p a r t containing the preamplifiers, which is installed inside the measuring cage, near to the electrode holder. Since the m e a s u r e d values m a y be affected b y DCpotentials of the brain, the polarization voltage is achieved b y a feedback-circuit which keeps the voltage c o n s t a n t b e t w e e n electrode and reference electrode, b y c o m p a r i n g the actual value w i t h a highly stabilized reference voltage. The difference b e t w e e n these 2 values m a y reach a m a x i m u m of 100 ~xV. This v o l t a g e - d r o p at the preamplifier i n p u t is negligible, so t h a t t h e current amplifier has an ideal i n p u t resistance of almost zero. Using F E T - i n p u t amplifiers, t h e whole current flows t h r o u g h the feedback-circuit and, b y o h m i c law, the o u t p u t voltage of the DC c u r r e n t - t o - v o l t a g e c o n v e r t e r circuit is proportional to t h e current in the polarographic system and the ohmic resistance in the feedback circuit. The capacitance of the preamplifier i n p u t is e x t r e m e l y low (3pf), the i n p u t resistance v e r y high (1012 Ohm). This enables 2 i n d e p e n d e n t feedback circuits (for AC and DC) to be c o n s t r u c t e d in t h e preamplifier wiring. Therefore, the capacitance in the i n p u t circuit is low and static charges in the polarographic circuit are avoided. For recording of A P s ( = AC signal), the preamplifier circuit works as a low noise AC-amplifier. H e r e b y t h e capacitance at the electrode tip is included in the wiring d i a g r a m of the

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first F E T - a m p l i f i e r . The AC-amplifier needs the elect r o d e c a p a c i t y for stable working. F o r this reason, the real i n p u t of t h e AC-amplifying system is the tissueside surface of the electrode m e m b r a n e , and so the length b e t w e e n measuring point and AC-amplifier i n p u t is zero. In this way, APs are recorded w i t h a good signal-tonoise ratio b y a d j u s t i n g the gain of t h e AC-amplifier. The following stages of amplifiers lead to a separation (by RC-coupling) into APs and pO~ channels. I n b o t h channels, t h e zero level and the amplification can be adjusted. F o r m o n i t o r i n g of APs, an optoelectronic light e m i t t e r (for visual control), an oscilloscope and loudspeakers are used. All amplifiers are of course supplied b y a highly stabilized voltage, while t h e reference voltage and the power s u p p l y for the preamplifier are s e p a r a t e l y stabilized. The whole system is supplied b y 2 high power 18V accumulators, which are charged when the system is switched off. C) Experimental application. I n Figure 2 a simultaneous recording of pO 2 and A P s is shown, as o b t a i n e d at a d e p t h of a p p r o x i m a t e l y 500 ~zm in the p a r i e t a l c o r t e x of cat (body w e i g h t 2.7 kg, N e m b u t a l 40 mg/kg). 20 sec after beginning of respiratory h y p o x i a (5% 03, 95% N~), the pOe drops and no sign of neuronal a c t i v i t y is seen after 20 sec. A f t e r 2 rain hypoxia, return to air b r e a t h i n g is accompanied b y a rapid increase of pO 2 which, following a period of overshoot, retursn to t h e original value w i t h i n a few minutes. The firing rate of the neurons rises progressively (Figure 2b) until it reaches the values before hypoxia, according to t h e findings of other authors 4,5. a H. I. BIGHER, D. F. BRULEY,D. D. R E N E A U and M. H. KNISLEY, in Bibliotheca Anatomica; Karger Verlag, Basel 1973), vol. 11, p. 526. 5 S. KUNKE, W. ERDMANNand H. METZGER,J. appl. Physiol. 32, 436 (1972).

F o r m a l d e h y d e - S c h i f f ' s R e a g e n t as a N u c l e o l a r Stain S. G H O S H

Chromosome Research Centre, Department o~ Botany, University o~ Calcutta, Calcutta 700079 (India), 7 October 7975. Summary. The use of f o r m a l d e h y d e - S c h i f f ' s reagent as a nucleolar stain has been described. Using different digestion procedures, it was confirmed t h a t the stain is specific for R N A . I t can be suitably used as a nucleolar stain, particularly in plant materials after a short TCA extraction, which p r o b a b l y extracts the n o n b o u n d R N A . The use of f o r m a l d e h y d e - S c h i f f ' s reagent as a nucleolar stain was reported in a previous c o m m u n i c a t i o n (GHosH 1). I n plant materials this procedure has distinct a d v a n t a g e s o v e r o t h e r k n o w n methods. In this note the specificity of the stain is presented. The stain is prepared b y adding dilute f o r m a l d e h y d e in drops to freshly prepared Schiff's reagent (DE TOMASI, cited in PEARSE 2) till the colour turns deep pink. The staining procedure has been described earlier (GHosI~l). A f t e r fixation, the m a t e r i a l is t r e a t e d w i t h 5% TCA at 90 ~ for 3 rain, washed and stained in the f o r m a l d e h y d e Schiff's reagent for 10-15 min, differentiated in 70% alcohol and squashed in a drop of 45% acetic acid. The specificity of the stain was i n v e s t i g a t e d in the Ehrlich mouse ascites turnout cells. Cell films were prepared and control preparations were stained as above. I n some preparations the TCA e x t r a c t i o n was o m i t t e d and the normaI staining procedure was followed. Different e x t r a c t i o n procedures were as follows : 1. E x t r a c t e d

in 10% perchloric acid at 4 ~ 20 h. W a s h e d t h o r o u g h l y in running w a t e r before staining. 2. Some preparations were treated w i t h R N a s e (COI W o r t h i n g t o n 13iochemicals) in p h o s p h a t e buffer (100v/ml, p H 7.2) for 2 h and stained as usual. 3. R N a s e t r e a t e d cells were e x t r a c t e d further as 1. 4. S o m e preparations were t r e a t e d w i t h DNase (free of RNase, Serva) in p h o s p h a t e buffer (1 mg/ml, p H 7.2) containing 0.003 M MgSO 4 for 20 h at 37~ 5. Others were e x t r a c t e d w i t h pronase (Calbiochem) in p h o s p h a t e buffer (100?/ml, p H 7.2) for 2 h. 6. Some preparations were t r e a t e d w i t h 5% TCA at 90~ for 25 min to e x t r a c t b o t h D N A and R N A and stained as usual. Figure 1 shows nucleolar staining in A. cepa roottip cells. Figure 2 represents cells in higher magnification showing nucteotar details. Staining of nucleoli can be seen I S. GHOSH, Naturwissenschaften 67, 687 (1974).

A. G. E. PEARSE,Theorctidal and Applied Histochemistry, 3rd edn. (Churchill, London 1968], vol. 1, p. 647.

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Specialia

EXPERIENTIA 32]5 Figs. 1-5. Nucleolar staining through formaldehyde-Sehiff's re-

agent. 1 and 2. Nucleoli in A. cepa roottip cells. 3. Mouse ascites tumour cells. 4 and 5. MAT cells treated with DNase and pronase respectively. 1, 3 and 5,• 2 and 4, • 1,500.

in c o n t r o l p r e p a r a t i o n s f r o m ascites ceils in F i g u r e 3. W'hen t h e TCA e x t r a c t i o n was o m i t t e d , t h e cells r e v e a l e d overall s t a i n i n g a n d p r o l o n g e d T C A e x t r a c t i o n r e s u l t e d in c o m p l e t e loss of s t a i n a b i l i t y . P r e p a r a t i o n s u n d e r g o i n g treatments 1 and 2 exhibited faint nucleolar and cytop l a s m i c staining, b u t no s t a i n was o b t a i n e d w h e r e R N a s e t r e a t m e n t was followed b y perchloric acid e x t r a c t i o n .

Holding Plastic-Embedded

P r e p a r a t i o n s f r o m 4 a n d 5 e x h i b i t e d o n l y s l i g h t decrease in s t a i n a b i l i t y (Figures 4 a n d 5). T h e r e s u l t s i n d i c a t e clearly t h a t t h e s t a i n is specific for R N A . T h e s h o r t T C A e x t r a c t i o n before s t a i n i n g is p e r h a p s n e e d e d t o e x t r a c t t h e n o n - b o u n d R N A a n d to o b t a i n a specific n u c l e o l a r stain. T h e free a m i n o g r o u p s of t h e p a r a r o s a n i l i n e molecules p e r h a p s r e a c t w i t h t h e n u c l e o l a r m a t e r i a l .

S p e c i m e n s for S e c t i o n i n g in a R o t a r y M i c r o t o m e 1

M. B. COLAS, JR.

Department o/ Orthopedic Surgery, Loyola Medical Center, 2150 South First Avenue, Maywood (Illinois 50153, USA), 70 November 7975. Summary. T h r e e m e t h o d s are e v a l u a t e d for h o l d i n g capsules of p l a s t i c - e m b e d d e d tissue for r o t a r y m i c r o t o m y . Use of a V - b l o c k is r a p i d b u t d e f o r m s t h e capsule. G l u i n g t h e capsule t o a s u p p o r t i n g b l o c k is useful for r e o r i e n t a t i o n of t h e capsule b u t o t h e r w i s e t i m e c o n s u m i n g . A m e t a l a d a p t e r is easy t o use, does n o t d e f o r m t h e tissue, a n d is t h e p r e f e r r e d m e t h o d for r o u t i n e m i c r o t o m y . Several l a b o r a t o r i e s use plastic to e m b e d biological s p e c i m e n s w h i c h m a y be s e c t i o n e d u s i n g a r o t a r y microt o m e 2-~2. To p r o d u c e q u a l i t y sections, e m b e d d e d tissue m u s t b e h e l d securely in t h e m i c r o t o m e o b j e c t clamp. T h i s is especially t r u e w h e n sections of 0.5 to 5.0 a m of p l a s t i c - e m b e d d e d tissue are cut. Since p l a s t i c - e m b e d d e d tissues are u s u a l l y c a s t in m o l d s of g e l a t i n or p o l y e t h y l e n e

capsules, t h e p r o b l e m arises of h o w to h o l d t h e s m a l l c y l i n d e r s of p l a s t i c - e m b e d d e d tissue in t h e o b j e c t c l a m p of a r o t a r y m i c r o t o m e w h i c h was d e s i g n e d to h o l d r e c t a n g u l a r blocks. R a t l i v e r was fixed in g h t a r a l d e h y d e , e m b e d d e d in glycol m e t h a c r y l a t e a n d c a s t in size 00 g e l a t i n capsules G. 3 m e t h o d s for h o l d i n g t h e p l a s t i c c y l i n d e r s in a n A m e r i -

Formaldehyde-Schiff's reagent as a nucleolar strain.

15.5. 1976 Specialia B) The recording system. As shown above, recording of pO 2 and APs m u s t be done at the same electrode tip. B o t h APs and p...
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