7.3.
ALKALINE LEAD HYDROXIDE STAINS
219
seem to require hours of soaking to achieve full intensity. With heat the
time may be reduced to 30 minutes or less.
In general it seems best to complete the staining of sections on the day
they are cut and mounted. Otherwise weak stains may result, perhaps
because of slow alterations in the embedment, which affect their penetration. Also, unsupported sections seem less likely to survive the hazards
of staining after prolonged standing, than if freshly made.
7.3. Alkaline Lead Hydroxide Stains
In 1958 Watson introduced a lead "hydroxide" stain for electron
microscopy which, with its variants, has proved to be by far the most
valuable general purpose stain. Its use, coupled with epoxy and polyester
embedments, made possible the dramatic strides in our knowledge of fine
structure during the 5 year period following its introduction. Indeed, it
is very difficult to imagine having to use these embedments without having
available the lead stains which provide the necessary contrast to visualize
most cytological features with unparalleled brilliance (compare Fig.
28(a) with 28(b)).
Reynolds (1963) discusses the mechanisms of staining with alkaline
lead salts and suggests that the fundamental mechanism of effective staining is similar in all instances, whether his stain is being used, or the
original formulation of Watson (1958), or other effective lead stains
introduced by Millonig (1961), Karnovsky (1961), and Lever (1960). He
suggests that divalent lead salts in highly alkaline solution probably form
compounds of the general type indicated, which ionize as shown in the
following equation:
Pb(OH)2 Pb X2 ^± [Pb(OH)2 Pb] + + + 2X"
There are theoretical and experimental reasons for believing that maximum concentrations of such polymeric cations would be present at
approximately pH 12, and Reynolds believes that such cations are the
effective staining agents. Their concentration falls off rapidly at higher
pH values as these complexes then are converted to anionic forms of
lead. Earlier suggestions as to the effective radical implicated anionic
complexes (Karnovsky, 1961).
From its inception, it was realized that Watson's stain had extraor-
ALKALINE LEAD HYDROXIDE STAINS
219
seem to require hours of soaking to achieve full intensity. With heat the
time may be reduced to 30 minutes or less.
In general it seems best to complete the staining of sections on the day
they are cut and mounted. Otherwise weak stains may result, perhaps
because of slow alterations in the embedment, which affect their penetration. Also, unsupported sections seem less likely to survive the hazards
of staining after prolonged standing, than if freshly made.
7.3. Alkaline Lead Hydroxide Stains
In 1958 Watson introduced a lead "hydroxide" stain for electron
microscopy which, with its variants, has proved to be by far the most
valuable general purpose stain. Its use, coupled with epoxy and polyester
embedments, made possible the dramatic strides in our knowledge of fine
structure during the 5 year period following its introduction. Indeed, it
is very difficult to imagine having to use these embedments without having
available the lead stains which provide the necessary contrast to visualize
most cytological features with unparalleled brilliance (compare Fig.
28(a) with 28(b)).
Reynolds (1963) discusses the mechanisms of staining with alkaline
lead salts and suggests that the fundamental mechanism of effective staining is similar in all instances, whether his stain is being used, or the
original formulation of Watson (1958), or other effective lead stains
introduced by Millonig (1961), Karnovsky (1961), and Lever (1960). He
suggests that divalent lead salts in highly alkaline solution probably form
compounds of the general type indicated, which ionize as shown in the
following equation:
Pb(OH)2 Pb X2 ^± [Pb(OH)2 Pb] + + + 2X"
There are theoretical and experimental reasons for believing that maximum concentrations of such polymeric cations would be present at
approximately pH 12, and Reynolds believes that such cations are the
effective staining agents. Their concentration falls off rapidly at higher
pH values as these complexes then are converted to anionic forms of
lead. Earlier suggestions as to the effective radical implicated anionic
complexes (Karnovsky, 1961).
From its inception, it was realized that Watson's stain had extraor-