Figure 1.
Pre-absorbtion of antibodies to mouse or human albumins.
In order to remove cross-reactivity between antibodies to human albumin and endogenous mouse albumin, antibodies to mouse albumin were pre-absorbed with human albumin and antibodies to human albumin were pre-absorbed with mouse albumin prior to immunocytochemical staining. A/A′. A section through mouse brain (A) and abdominal cavity (A′) stained with antibodies to mouse albumin. Note strong, wide spread positive immunoreactivity in the intestine, while positive reactivity in the brain is only present in choroid plexus and blood vessels of the cortex. B/B′. Similar sections stained with antibodies to human albumin illustrating the level of cross-reactivity. C/C′. Section from the same animal immunostained with antibodies to mouse albumin pre-absorbed with human albumin. Note that the positive staining is reduced compared to un-absorbed antibody (A) but is still visible. However when anti-human albumin antibodies are pre-absorbed with mouse albumin (D/D′), all staining is removed (compare D with B). E&F. Results from in situ PLA assay of the adult choroid plexus using antibodies to human albumin pre-absorbed with mouse albumin on sections from human albumin injected mouse (E) and saline injected control animal (F). These figures illustrate that the pre-absorption protocol used (see Methods) was adequate to remove any cross-reactivity between the two albumins both at the immunocytochemical and in situ PLA levels. G. Additional control – GFAP immunoreactivity in P10 mouse brain and choroid plexus. Note that choroidal tissue is devoid of positive staining. Abbreviations: Alb., albumin; cp, choroid plexus; Hu., human; Ms., mouse. Scale bar is 50µm in A–D′, G; 20µm in E–F.
Figure 2.
CSF/Plasma concentration ratios and Sparc expression in control and experimental mice.
A. The method of Bradford (1976) was used to detect the total protein concentration of CSF and plasma samples taken from control animals, and those injected with human albumin for 24 hours. These data are represented as CSF/plasma concentration ratios (%). *, p<0.05, compared to age-matched control. Abbreviation: HSA, human serum albumin. Actual protein concentrations are presented in Table 1. B. Western blot analysis of SPARC and albumin in plasma of control mice during development. This analysis was performed 3 times on separately pooled samples. For SPARC 1 µg total protein was loaded in each lane; for albumin 0.5 µg total protein was loaded in each lane. Note that SPARC levels increase from E15 until P10 and decline in adults. C. Relative expression of Sparc in the lateral ventricular choroid plexus of control mice. Expression at P2 was taken as 1. n = 4 pooled biological samples (with 4 technical replicates each) in each instance. * p<0.05, one-way ANOVA compared to P2. Note developmental decline in the expression levels. D. Relative expression of Sparc in the lateral ventricular choroid plexus 24 hours following i.p. injections of human serum albumin. Animals were either injected (i.p.) with 0.9% w/v NaCl sterile saline (control) or human albumin at 50 µg/g or 250 µg/g. Relative expression refers to the expression of the gene in relation to age-matched controls. n = 4 pooled biological samples (with 4 technical replicates each) in each instance. * p<0.05, One-way analysis of variance with Kruskal–Wallis post hoc test, compared to age-matched control. Note a significant decline at P2 and an increase at P10 but only after the higher dose of exogenous albumin.
Table 1.
Concentrations of total protein and albumins in CSF and Plasma of control and human albumin-injected postnatal mice.
Figure 3.
Overall tissue distribution of mouse albumin/SPARC.
The distribution is illustrated using the in situ Proximity Ligation Assay (PLA); signals (red/brown dots) indicate close molecular proximity of albumin and SPARC in the lateral ventricular choroid plexus at P2 (A), P10 (B) and adult (C) and of human serum albumin/SPARC in situ PLA signals at P2 (D), P10 (E) and adult (F). In A note that some plexus epithelial cells outlined by boxes and plasma in blood vessels encircled by ovals show a discrete signal. B shows very strong staining at P10 both in epithelial cells, some of which are outlined by boxes and plasma in blood vessels, some surrounded by ovals. Some positive signals in the CSF were also detected (arrowheads in 3B). C is from an adult plexus where it was still possible to detect positive signals in many plexus cells, some of which are outlined by boxes and in blood vessels (outlined by ovals) at a higher intensity than at P2 but definitely at a lower intensity than at P10. In D (P2), E (P10) and F (adult) human serum albumin/SPARC in situ PLA signals were in general absent from the CSF and plexus epithelial cells (boxes), but were present in very low amounts in ependymal blood vessels (marked with oval) at P10 and in plexus blood vessels to some extent (ovals) in adult animals. CSF, cerebrospinal fluid. Asterisks indicate the ependymal lining of the lateral ventricle. A–F, same magnification, scale bar is 50 µm.
Figure 4.
Cellular distribution of mouse albumin/SPARC.
(A–C) and human albumin/SPARC (D–F) in situ PLA signals in the lateral ventricular choroid plexus at P2 (A,D), P10 (B,E) and adult (C,F). Note at P2 that most of the signal was distributed within blood vessels (BV), often associated with red blood cells. Under this magnification it is possible to distinguish positive signals distributed in the basolateral cytoplasm of choroid plexus epithelial cells (arrows). In contrast to the mouse albumin/SPARC signal, the human albumin/SPARC signal (D) was very rarely found and nearly always only associated with blood vessels (BV). Only one positive signal was found and it appears to be located in the extended extracellular space (arrowhead). At P10 (B and E) a very strong signal was visible for mouse albumin/SPARC (B) in many plexus cells distributed throughout the whole cytoplasm, blood vessels (BV) and also in the CSF. The human albumin/SPARC signal (E) was generally only present in blood vessels (BV) but a very occasional signal was detected in the apparent extended extracellular space (arrowhead). The CSF space was negative. In the adult (C and F) a mouse albumin/SPARC signal was distributed clearly throughout the cytoplasm of some choroid plexus epithelial cells (one cell marked with an asterisk). The positive signal was also detected in the ependymal (EP) and sub-ependymal layers of the brain. The human albumin/SPARC signal (F) was visible in blood vessels (BV) but not in the CSF and only very sporadically in the plexus epithelium (two positive red dots are indicated by arrows). Otherwise plexus epithelial cells (boxes) showed no in situ PLA signal. CSF, cerebrospinal fluid, EP, ependymal, BV, blood vessels. Same magnification, scale bar is 20 µm.
Figure 5.
Subcellular distribution of mouse serum albumin/SPARC in situ PLA signals in the lateral ventricular choroid plexus epithelial cells in the adult.
The same section is shown in three different planes of focus (A, B and C) to illustrate the 3-dimentional distribution of the signals within seven different epithelial cells (1–7). Note that no positive signal was visible in cells 1 and 3, cells 2 and 6 were completely filled by the reaction product while cells 4 and 5 showed some positive signals. Up to a certain density of in situ PLA signals, they appeared as discreet dots (cell 4 and 6 in B), whereas at higher densities the signals coalesce and appear as a diffuse label in the cytoplasm (cell 2 in A and C). In the optimal plane of focus (cell 2 in B) he signals seem to form a branching, continuous transcellular tubular system filled with the reaction product. CSF, cerebrospinal fluid; EP, ependyma; BV, blood vessel. A–C, same magnification, scale bar is 20 µm.